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Sample records for 2a receptor a2ar

  1. Binding of the Antagonist Caffeine to the Human Adenosine Receptor hA2AR in Nearly Physiological Conditions

    PubMed Central

    Cao, Ruyin; Rossetti, Giulia; Bauer, Andreas; CarIoni, Paolo

    2015-01-01

    Lipid composition may significantly affect membrane proteins function, yet its impact on the protein structural determinants is not well understood. Here we present a comparative molecular dynamics (MD) study of the human adenosine receptor type 2A (hA2AR) in complex with caffeine—a system of high neuro-pharmacological relevance—within different membrane types. These are POPC, mixed POPC/POPE and cholesterol-rich membranes. 0.8-μs MD simulations unambiguously show that the helical folding of the amphipathic helix 8 depends on membrane contents. Most importantly, the distinct cholesterol binding into the cleft between helix 1 and 2 stabilizes a specific caffeine-binding pose against others visited during the simulation. Hence, cholesterol presence (~33%-50% in synaptic membrane in central nervous system), often neglected in X-ray determination of membrane proteins, affects the population of the ligand binding poses. We conclude that including a correct description of neuronal membranes may be very important for computer-aided design of ligands targeting hA2AR and possibly other GPCRs. PMID:25992797

  2. Binding of the Antagonist Caffeine to the Human Adenosine Receptor hA2AR in Nearly Physiological Conditions.

    PubMed

    Cao, Ruyin; Rossetti, Giulia; Bauer, Andreas; CarIoni, Paolo

    2015-01-01

    Lipid composition may significantly affect membrane proteins function, yet its impact on the protein structural determinants is not well understood. Here we present a comparative molecular dynamics (MD) study of the human adenosine receptor type 2A (hA(2A)R) in complex with caffeine--a system of high neuro-pharmacological relevance--within different membrane types. These are POPC, mixed POPC/POPE and cholesterol-rich membranes. 0.8-μs MD simulations unambiguously show that the helical folding of the amphipathic helix 8 depends on membrane contents. Most importantly, the distinct cholesterol binding into the cleft between helix 1 and 2 stabilizes a specific caffeine-binding pose against others visited during the simulation. Hence, cholesterol presence (~33%-50% in synaptic membrane in central nervous system), often neglected in X-ray determination of membrane proteins, affects the population of the ligand binding poses. We conclude that including a correct description of neuronal membranes may be very important for computer-aided design of ligands targeting hA(2A)R and possibly other GPCRs.

  3. Basal adenosine modulates the functional properties of AMPA receptors in mouse hippocampal neurons through the activation of A1R A2AR and A3R

    PubMed Central

    Di Angelantonio, Silvia; Bertollini, Cristina; Piccinin, Sonia; Rosito, Maria; Trettel, Flavia; Pagani, Francesca; Limatola, Cristina; Ragozzino, Davide

    2015-01-01

    Adenosine is a widespread neuromodulator within the CNS and its extracellular level is increased during hypoxia or intense synaptic activity, modulating pre- and postsynaptic sites. We studied the neuromodulatory action of adenosine on glutamatergic currents in the hippocampus, showing that activation of multiple adenosine receptors (ARs) by basal adenosine impacts postsynaptic site. Specifically, the stimulation of both A1R and A3R reduces AMPA currents, while A2AR has an opposite potentiating effect. The effect of ARs stimulation on glutamatergic currents in hippocampal cultures was investigated using pharmacological and genetic approaches. A3R inhibition by MRS1523 increased GluR1-Ser845 phosphorylation and potentiated AMPA current amplitude, increasing the apparent affinity for the agonist. A similar effect was observed blocking A1R with DPCPX or by genetic deletion of either A3R or A1R. Conversely, impairment of A2AR reduced AMPA currents, and decreased agonist sensitivity. Consistently, in hippocampal slices, ARs activation by AR agonist NECA modulated glutamatergic current amplitude evoked by AMPA application or afferent fiber stimulation. Opposite effects of AR subtypes stimulation are likely associated to changes in GluR1 phosphorylation and represent a novel mechanism of physiological modulation of glutamatergic transmission by adenosine, likely acting in normal conditions in the brain, depending on the level of extracellular adenosine and the distribution of AR subtypes. PMID:26528137

  4. Adenosine A2A Receptors and A2A Receptor Heteromers as Key Players in Striatal Function

    PubMed Central

    Ferré, Sergi; Quiroz, César; Orru, Marco; Guitart, Xavier; Navarro, Gemma; Cortés, Antonio; Casadó, Vicent; Canela, Enric I.; Lluis, Carme; Franco, Rafael

    2011-01-01

    A very significant density of adenosine A2A receptors (A2ARs) is present in the striatum, where they are preferentially localized postsynaptically in striatopallidal medium spiny neurons (MSNs). In this localization A2ARs establish reciprocal antagonistic interactions with dopamine D2 receptors (D2Rs). In one type of interaction, A2AR and D2R are forming heteromers and, by means of an allosteric interaction, A2AR counteracts D2R-mediated inhibitory modulation of the effects of NMDA receptor stimulation in the striatopallidal neuron. This interaction is probably mostly responsible for the locomotor depressant and activating effects of A2AR agonist and antagonists, respectively. The second type of interaction involves A2AR and D2R that do not form heteromers and takes place at the level of adenylyl cyclase (AC). Due to a strong tonic effect of endogenous dopamine on striatal D2R, this interaction keeps A2AR from signaling through AC. However, under conditions of dopamine depletion or with blockade of D2R, A2AR-mediated AC activation is unleashed with an increased gene expression and activity of the striatopallidal neuron and with a consequent motor depression. This interaction is probably the main mechanism responsible for the locomotor depression induced by D2R antagonists. Finally, striatal A2ARs are also localized presynaptically, in cortico-striatal glutamatergic terminals that contact the striato-nigral MSN. These presynaptic A2ARs heteromerize with A1 receptors (A1Rs) and their activation facilitates glutamate release. These three different types of A2ARs can be pharmacologically dissected by their ability to bind ligands with different affinity and can therefore provide selective targets for drug development in different basal ganglia disorders. PMID:21731559

  5. Dissecting striatal adenosine-cannabinoid receptor interactions. New clues from rats over-expressing adenosine A2A receptors.

    PubMed

    Ferré, Sergi; Sebastião, Ana Maria

    2016-03-01

    This Editorial highlights a study by Chiodi et al. () showing that the effects mediated by cannabinoid CB1 receptor (CB1R) activation in the striatum are significantly reduced in rats with neuronal over-expression of adenosine A2A receptors (A2AR). Two hypotheses are derived from that study. Hypothesis A: two subpopulations of pre-synaptic CB1R in corticostriatal glutamatergic terminals exist, one forming and another not forming heteromers with A2AR. Hypothesis B: CB1R are predominantly forming heteromers with A2AR. In the case of hypothesis A, the A2AR might be required for CB1R-A2AR heteromeric signaling, whereas non-heteromeric CB1R activity is inhibited by A2ARs. In the case of hypothesis B, up-regulation of A2ARs may perturb heteromeric stoichiometry, thus reducing CB1R functioning. In any case, pre-synaptic striatal A2AR-CB1R heteromers emerge as important targets of the effects of cannabinoids demonstrated at the neuronal and behavioral level. Read the highlighted article 'Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors' on page 907.

  6. Calcium modulates calmodulin/α-actinin 1 interaction with and agonist-dependent internalization of the adenosine A2A receptor.

    PubMed

    Piirainen, Henni; Taura, Jaume; Kursula, Petri; Ciruela, Francisco; Jaakola, Veli-Pekka

    2017-04-01

    Adenosine receptors are G protein-coupled receptors that sense extracellular adenosine to transmit intracellular signals. One of the four adenosine receptor subtypes, the adenosine A2A receptor (A2AR), has an exceptionally long intracellular C terminus (A2AR-ct) that mediates interactions with a large array of proteins, including calmodulin and α-actinin. Here, we aimed to ascertain the α-actinin 1/calmodulin interplay whilst binding to A2AR and the role of Ca(2+) in this process. First, we studied the A2AR-α-actinin 1 interaction by means of native polyacrylamide gel electrophoresis, isothermal titration calorimetry, and surface plasmon resonance, using purified recombinant proteins. α-Actinin 1 binds the A2AR-ct through its distal calmodulin-like domain in a Ca(2+)-independent manner with a dissociation constant of 5-12μM, thus showing an ~100 times lower affinity compared to the A2AR-calmodulin/Ca(2+) complex. Importantly, calmodulin displaced α-actinin 1 from the A2AR-ct in a Ca(2+)-dependent fashion, disrupting the A2AR-α-actinin 1 complex. Finally, we assessed the impact of Ca(2+) on A2AR internalization in living cells, a function operated by the A2AR-α-actinin 1 complex. Interestingly, while Ca(2+) influx did not affect constitutive A2AR endocytosis, it abolished agonist-dependent internalization. In addition, we demonstrated that the A2AR/α-actinin interaction plays a pivotal role in receptor internalization and function. Overall, our results suggest that the interplay of A2AR with calmodulin and α-actinin 1 is fine-tuned by Ca(2+), a fact that might power agonist-mediated receptor internalization and function.

  7. Adenosine 2A receptor agonism: A single intrathecal administration attenuates motor paralysis in experimental autoimmune encephalopathy in rats.

    PubMed

    Loram, Lisa C; Strand, Keith A; Taylor, Frederick R; Sloane, Evan; Van Dam, Anne-Marie; Rieger, Jayson; Maier, Steven F; Watkins, Linda R

    2015-05-01

    A single intrathecal dose of adenosine 2A receptor (A2AR) agonist was previously reported to produce a multi-week reversal of allodynia in two different models of neuropathic pain in addition to downregulating glial activation markers in the spinal cord. We aimed to determine whether a single intrathecal administration of an A2AR agonist was able to attenuate motor symptoms induced by experimental autoimmune encephalopathy. Two A2AR agonists (CGS21680 and ATL313) significantly attenuated progression of motor symptoms following a single intrathecal administration at the onset of motor symptoms. OX-42, a marker of microglial activation, was significantly attenuated in the lumbar spinal cord following A2AR administration compared to vehicle. Therefore, A2AR agonists attenuate motor symptoms of EAE by acting on A2AR in the spinal cord.

  8. Habit Formation after Random Interval Training Is Associated with Increased Adenosine A2A Receptor and Dopamine D2 Receptor Heterodimers in the Striatum

    PubMed Central

    He, Yan; Li, Yan; Chen, Mozi; Pu, Zhilan; Zhang, Feiyang; Chen, Long; Ruan, Yang; Pan, Xinran; He, Chaoxiang; Chen, Xingjun; Li, Zhihui; Chen, Jiang-Fan

    2016-01-01

    Striatal adenosine A2A receptors (A2ARs) modulate striatal synaptic plasticity and instrumental learning, possibly by functional interaction with the dopamine D2 receptors (D2Rs) and metabotropic glutamate receptors 5 (mGluR5) through receptor-receptor heterodimers, but in vivo evidence for these interactions is lacking. Using in situ proximity ligation assay (PLA), we studied the subregional distribution of the A2AR-D2R and A2AR-mGluR5 heterodimer complexes in the striatum and their adaptive changes over the random interval and random ratio training of instrumental learning. After confirming the specificity of the PLA detection of the A2AR-D2R heterodimers with the A2AR knockout and D2R knockout mice, we detected a heterogeneous distribution of the A2AR-D2R heterodimer complexes in the striatum, being more abundant in the dorsolateral than the dorsomedial striatum. Importantly, habit formation after the random interval training was associated with the increased formation of the A2AR-D2R heterodimer complexes, with prominant increase in the dorsomedial striatum. Conversely, goal-directed behavior after the random ratio schedule was not associated with the adaptive change in the A2AR-D2R heterodimer complexes. In contrast to the A2AR-D2R heterodimers, the A2AR-mGluR5 heterodimers showed neither subregional variation in the striatum nor adaptive changes over either the random ratio (RR) or random interval (RI) training of instrumental learning. These findings suggest that development of habit formation is associated with increased formation of the A2AR-D2R heterodimer protein complexes which may lead to reduced dependence on D2R signaling in the striatum. PMID:28082865

  9. MC5r and A2Ar Deficiencies During Experimental Autoimmune Uveitis Identifies Distinct T cell Polarization Programs and a Biphasic Regulatory Response.

    PubMed

    Lee, Darren J; Preble, Janine; Lee, Stacey; Foster, C Stephen; Taylor, Andrew W

    2016-11-25

    Autoantigen-specific regulatory immunity emerges in the spleen of mice recovering from experimental autoimmune uveitis (EAU), a murine model for human autoimmune uveoretinitis. This regulatory immunity provides induced tolerance to ocular autoantigen, and requires melanocortin 5 receptor (MC5r) expression on antigen presenting cells with adenosine 2 A receptor (A2Ar) expression on T cells. During EAU it is not well understood what roles MC5r and A2Ar have on promoting regulatory immunity. Cytokine profile analysis during EAU revealed MC5r and A2Ar each mediate distinct T cell responses, and are responsible for a functional regulatory immune response in the spleen. A2Ar stimulation at EAU onset did not augment this regulatory response, nor bypass the MC5r requirement to induce regulatory immunity. The importance of this pathway in human autoimmune uveitis was assayed. PBMC from uveitis patients were assayed for MC5r expression on monocytes and A2Ar on T cells, and comparison between uveitis patients and healthy controls had no significant difference. The importance for MC5r and A2Ar expression in EAU to promote the induction of protective regulatory immunity, and the expression of MC5r and A2Ar on human immune cells, suggests that it may be possible to utilize the melanocortin-adenosinergic pathways to induce protective immunity in uveitic patients.

  10. MC5r and A2Ar Deficiencies During Experimental Autoimmune Uveitis Identifies Distinct T cell Polarization Programs and a Biphasic Regulatory Response

    PubMed Central

    Lee, Darren J.; Preble, Janine; Lee, Stacey; Foster, C. Stephen; Taylor, Andrew W.

    2016-01-01

    Autoantigen-specific regulatory immunity emerges in the spleen of mice recovering from experimental autoimmune uveitis (EAU), a murine model for human autoimmune uveoretinitis. This regulatory immunity provides induced tolerance to ocular autoantigen, and requires melanocortin 5 receptor (MC5r) expression on antigen presenting cells with adenosine 2 A receptor (A2Ar) expression on T cells. During EAU it is not well understood what roles MC5r and A2Ar have on promoting regulatory immunity. Cytokine profile analysis during EAU revealed MC5r and A2Ar each mediate distinct T cell responses, and are responsible for a functional regulatory immune response in the spleen. A2Ar stimulation at EAU onset did not augment this regulatory response, nor bypass the MC5r requirement to induce regulatory immunity. The importance of this pathway in human autoimmune uveitis was assayed. PBMC from uveitis patients were assayed for MC5r expression on monocytes and A2Ar on T cells, and comparison between uveitis patients and healthy controls had no significant difference. The importance for MC5r and A2Ar expression in EAU to promote the induction of protective regulatory immunity, and the expression of MC5r and A2Ar on human immune cells, suggests that it may be possible to utilize the melanocortin-adenosinergic pathways to induce protective immunity in uveitic patients. PMID:27886238

  11. MOLECULAR DETERMINANTS OF A2AR-D2R ALLOSTERISM: ROLE OF THE INTRACELLULAR LOOP 3 OF THE D2R

    PubMed Central

    Fernández-Dueñas, Víctor; Gómez-Soler, Maricel; Jacobson, Kenneth A.; Kumar, T. Santhosh; Fuxe, Kjell; Borroto-Escuela, Dasiel O.; Ciruela, Francisco

    2012-01-01

    In the central nervous system (CNS), an antagonistic interaction has been shown between adenosine A2A and dopamine D2 receptors (A2ARs and D2Rs) that may be relevant both in normal and pathological conditions (i.e. Parkinson’s disease). Thus, the molecular determinants mediating this receptor-receptor interaction have recently been explored, since the fine tuning of this target (namely the A2AR/D2R oligomer) could possibly improve the treatment of certain CNS diseases. Here, we used a fluorescence resonance energy transfer (FRET)-based approach to examine the allosteric modulation of the D2R within the A2AR/D2R oligomer and the dependence of this receptor-receptor interaction on two regions rich in positive charges on intracellular loop 3 (IL3) of the D2R. Interestingly, we observed a negative allosteric effect of the D2R agonist quinpirole on A2AR ligand binding and activation. However, these allosteric effects were abolished upon mutation of specific arginine residues (217–222 and 267–269) on IL3 of the D2R, thus demonstrating a major role of these positively-charged residues in mediating the observed receptor-receptor interaction. Overall, these results provide structural insights to better understand the functioning of the A2AR/D2R oligomer in living cells. PMID:22924752

  12. The caffeine-binding adenosine A2A receptor induces age-like HPA-axis dysfunction by targeting glucocorticoid receptor function.

    PubMed

    Batalha, Vânia L; Ferreira, Diana G; Coelho, Joana E; Valadas, Jorge S; Gomes, Rui; Temido-Ferreira, Mariana; Shmidt, Tatiana; Baqi, Younis; Buée, Luc; Müller, Christa E; Hamdane, Malika; Outeiro, Tiago F; Bader, Michael; Meijsing, Sebastiaan H; Sadri-Vakili, Ghazaleh; Blum, David; Lopes, Luísa V

    2016-08-11

    Caffeine is associated with procognitive effects in humans by counteracting overactivation of the adenosine A2A receptor (A2AR), which is upregulated in the human forebrain of aged and Alzheimer's disease (AD) patients. We have previously shown that an anti-A2AR therapy reverts age-like memory deficits, by reestablishment of the hypothalamic-pituitary-adrenal (HPA) axis feedback and corticosterone circadian levels. These observations suggest that A2AR over-activation and glucocorticoid dysfunction are key events in age-related hippocampal deficits; but their direct connection has never been explored. We now show that inducing A2AR overexpression in an aging-like profile is sufficient to trigger HPA-axis dysfunction, namely loss of plasmatic corticosterone circadian oscillation, and promotes reduction of GR hippocampal levels. The synaptic plasticity and memory deficits triggered by GR in the hippocampus are amplified by A2AR over-activation and were rescued by anti-A2AR therapy; finally, we demonstrate that A2AR act on GR nuclear translocation and GR-dependent transcriptional regulation. We provide the first demonstration that A2AR is a major regulator of GR function and that this functional interconnection may be a trigger to age-related memory deficits. This supports the idea that the procognitive effects of A2AR antagonists, namely caffeine, on Alzheimer's and age-related cognitive impairments may rely on its ability to modulate GR actions.

  13. The caffeine-binding adenosine A2A receptor induces age-like HPA-axis dysfunction by targeting glucocorticoid receptor function

    PubMed Central

    Batalha, Vânia L.; Ferreira, Diana G.; Coelho, Joana E.; Valadas, Jorge S.; Gomes, Rui; Temido-Ferreira, Mariana; Shmidt, Tatiana; Baqi, Younis; Buée, Luc; Müller, Christa E.; Hamdane, Malika; Outeiro, Tiago F.; Bader, Michael; Meijsing, Sebastiaan H.; Sadri-Vakili, Ghazaleh; Blum, David; Lopes, Luísa V.

    2016-01-01

    Caffeine is associated with procognitive effects in humans by counteracting overactivation of the adenosine A2A receptor (A2AR), which is upregulated in the human forebrain of aged and Alzheimer’s disease (AD) patients. We have previously shown that an anti-A2AR therapy reverts age-like memory deficits, by reestablishment of the hypothalamic-pituitary-adrenal (HPA) axis feedback and corticosterone circadian levels. These observations suggest that A2AR over-activation and glucocorticoid dysfunction are key events in age-related hippocampal deficits; but their direct connection has never been explored. We now show that inducing A2AR overexpression in an aging-like profile is sufficient to trigger HPA-axis dysfunction, namely loss of plasmatic corticosterone circadian oscillation, and promotes reduction of GR hippocampal levels. The synaptic plasticity and memory deficits triggered by GR in the hippocampus are amplified by A2AR over-activation and were rescued by anti-A2AR therapy; finally, we demonstrate that A2AR act on GR nuclear translocation and GR-dependent transcriptional regulation. We provide the first demonstration that A2AR is a major regulator of GR function and that this functional interconnection may be a trigger to age-related memory deficits. This supports the idea that the procognitive effects of A2AR antagonists, namely caffeine, on Alzheimer’s and age-related cognitive impairments may rely on its ability to modulate GR actions. PMID:27510168

  14. Neuroprotection by caffeine in the MPTP model of parkinson's disease and its dependence on adenosine A2A receptors.

    PubMed

    Xu, K; Di Luca, D G; Orrú, M; Xu, Y; Chen, J-F; Schwarzschild, M A

    2016-05-13

    Considerable epidemiological and laboratory data have suggested that caffeine, a nonselective adenosine receptor antagonist, may protect against the underlying neurodegeneration of parkinson's disease (PD). Although both caffeine and more specific antagonists of the A2A subtype of adenosine receptor (A2AR) have been found to confer protection in animal models of PD, the dependence of caffeine's neuroprotective effects on the A2AR is not known. To definitively determine its A2AR dependence, the effect of caffeine on 1-methyl-4-phenyl-1,2,3,6 tetra-hydropyridine (MPTP) neurotoxicity was compared in wild-type (WT) and A2AR gene global knockout (A2A KO) mice, as well as in central nervous system (CNS) cell type-specific (conditional) A2AR knockout (cKO) mice that lack the receptor either in postnatal forebrain neurons or in astrocytes. In WT and in heterozygous A2AR KO mice caffeine pretreatment (25mg/kgip) significantly attenuated MPTP-induced depletion of striatal dopamine. By contrast in homozygous A2AR global KO mice caffeine had no effect on MPTP toxicity. In forebrain neuron A2AR cKO mice, caffeine lost its locomotor stimulant effect, whereas its neuroprotective effect was mostly preserved. In astrocytic A2AR cKO mice, both caffeine's locomotor stimulant and protective properties were undiminished. Taken together, these results indicate that neuroprotection by caffeine in the MPTP model of PD relies on the A2AR, although the specific cellular localization of these receptors remains to be determined.

  15. GPR88 in A2AR Neurons Enhances Anxiety-Like Behaviors

    PubMed Central

    Meirsman, Aura Carole; Robé, Anne

    2016-01-01

    Abstract GPR88 is an orphan G-protein-coupled receptor highly expressed in striatal dopamine D1 (receptor) R- and D2R-expressing medium spiny neurons. This receptor is involved in activity and motor responses, and we previously showed that this receptor also regulates anxiety-like behaviors. To determine whether GPR88 in D2R-expressing neurons contributes to this emotional phenotype, we generated conditional Gpr88 knock-out mice using adenosine A2AR (A2AR)-Cre-driven recombination, and compared anxiety-related responses in both total and A2AR-Gpr88 KO mice. A2AR-Gpr88 KO mice showed a selective reduction of Gpr88 mRNA in D2R-expressing, but not D1R-expressing, neurons. These mutant mice showed increased locomotor activity and decreased anxiety-like behaviors in light/dark and elevated plus maze tests. These phenotypes were superimposable on those observed in total Gpr88 KO mice, demonstrating that the previously reported anxiogenic activity of GPR88 operates at the level of A2AR-expressing neurons. Further, A2AR-Gpr88 KO mice showed no change in novelty preference and novelty-suppressed feeding, while these responses were increased and decreased, respectively, in the total Gpr88 KO mice. Also, A2AR-Gpr88 KO mice showed intact fear conditioning, while the fear responses were decreased in total Gpr88 KO. We therefore also show for the first time that GPR88 activity regulates approach behaviors and conditional fear; however, these behaviors do not seem mediated by receptors in A2AR neurons. We conclude that Gpr88 expressed in A2AR neurons enhances ethological anxiety-like behaviors without affecting conflict anxiety and fear responses. PMID:27570825

  16. Therapeutic Opportunities for Caffeine and A2A Receptor Antagonists in Retinal Diseases.

    PubMed

    Boia, Raquel; Ambrósio, António Francisco; Santiago, Ana Raquel

    2016-01-01

    Caffeine, the major component of coffee, is the most consumed psychostimulant in the world. Caffeine is an adenosine analog and acts as a nonselective adenosine receptor antagonist. The majority of the effects of caffeine are mainly mediated by the blockade of adenosine receptors, and the proved neuroprotective effects of caffeine in brain disorders have been mimicked by the blockade of adenosine A2A receptor (A2AR). A growing body of evidence demonstrates that microglia-mediated neuroinflammation plays a key role in the pathophysiology of brain and retinal diseases. Moreover, the control of microglia reactivity by blocking A2AR has been proposed to be the mechanism underlying the observed protective effects of caffeine. Hence, it is conceivable that caffeine and A2AR antagonists offer therapeutic value for the treatment of retinal diseases, mainly those involving microglia-mediated neuroinflammation.

  17. Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket

    PubMed Central

    Sun, Bingfa; Bachhawat, Priti; Chu, Matthew Ling-Hon; Wood, Martyn; Ceska, Tom; Sands, Zara A.; Mercier, Joel; Lebon, Florence; Kobilka, Tong Sun; Kobilka, Brian K.

    2017-01-01

    The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease. PMID:28167788

  18. Adenosine A2A receptor signaling attenuates LPS-induced pro-inflammatory cytokine formation of mouse macrophages by inducing the expression of DUSP1.

    PubMed

    Köröskényi, Krisztina; Kiss, Beáta; Szondy, Zsuzsa

    2016-07-01

    Adenosine is known to reduce inflammation by suppressing the activity of most immune cells. Previous studies have shown that lipopolysaccharide (LPS) stimulated mouse macrophages produce adenosine, and the adenosine A2A receptor (A2AR) signaling activated in an autocrine manner attenuates LPS-induced pro-inflammatory cytokine formation. It has been suggested that A2AR signaling inhibits LPS-induced pro-inflammatory cytokine production through a unique cAMP-dependent, but PKA- and Epac-independent signaling pathway. However, the mechanism of inhibition was not identified so far. Here we report that LPS stimulation enhances A2AR expression in mouse bone marrow derived macrophages, and loss of A2ARs results in enhanced LPS-induced pro-inflammatory response. Loss of A2ARs in A2AR null macrophages did not alter the LPS-induced NF-κB activation, but an enhanced basal and LPS-induced phosphorylation of MAP kinases (especially that of JNKs) was detected in A2AR null cells. A2AR signaling did not alter the LPS-induced phosphorylation of their upstream kinases, but by regulating adenylate cyclase activity it enhanced the expression of dual specific phosphatase (DUSP)1, a negative regulator of MAP kinases. As a result, lower basal and LPS-induced DUSP1 mRNA and protein levels can be detected in A2AR null macrophages. Silencing of DUSP1 mRNA expression resulted in higher basal and LPS-induced JNK phosphorylation and LPS-induced pro-inflammatory cytokine formation in wild type macrophages, but had no effect on that in A2AR null cells. Our data indicate that A2AR signaling regulates both basal and LPS-induced DUSP1 levels in macrophages via activating the adenylate cyclase pathway.

  19. Reduced striatal adenosine A2A receptor levels define a molecular subgroup in schizophrenia.

    PubMed

    Villar-Menéndez, Izaskun; Díaz-Sánchez, Sara; Blanch, Marta; Albasanz, José Luis; Pereira-Veiga, Thais; Monje, Alfonso; Planchat, Luis Maria; Ferrer, Isidre; Martín, Mairena; Barrachina, Marta

    2014-04-01

    Schizophrenia (SZ) is a mental disorder of unknown origin. Some scientific evidence seems to indicate that SZ is not a single disease entity, since there are patient groups with clear symptomatic, course and biomarker differences. SZ is characterized by a hyperdopaminergic state related to high dopamine D2 receptor activity. It has also been proposed that there is a hypoadenosynergic state. Adenosine is a nucleoside widely distributed in the organism with neuromodulative and neuroprotective activity in the central nervous system. In the brain, the most abundant adenosine receptors are A1R and A2AR. In the present report, we characterize the presence of both receptors in human postmortem putamens of patients suffering SZ with real time TaqMan PCR, western blotting and radioligand binding assay. We show that A1R levels remain unchanged with respect to age-matched controls, whereas nearly fifty percent of patients have reduced A2AR, at the transcriptional and translational levels. Moreover, we describe how DNA methylation plays a role in the pathological A2AR levels with the bisulfite-sequencing technique. In fact, an increase in 5-methylcytosine percentage in the 5' UTR region of ADORA2A was found in those SZ patients with reduced A2AR levels. Interestingly, there was a relationship between the A2A/β-actin ratio and motor disturbances as assessed with some items of the PANSS, AIMS and SAS scales. Therefore, there may be a subgroup of SZ patients with reduced striatal A2AR levels accompanied by an altered motor phenotype.

  20. Chronic oral administration of MPEP, an antagonist of mGlu5 receptor, during gestation and lactation alters mGlu5 and A2A receptors in maternal and neonatal brain.

    PubMed

    López-Zapata, Antonio; León-Navarro, David Agustín; Crespo, María; Albasanz, José Luis; Martín, Mairena

    2017-03-06

    Antidepressant and anxiolytic drugs are widely consumed even by pregnant and lactating women. The metabotropic glutamate receptor 5 (mGlu5) antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) exerts antidepressant- and anxiolytic-like actions. Given that treatment for anxiety and depression use to be prolonged in time, it is conceivable a possible modulation of metabotropic glutamate receptors (mGlu receptors) after prolonged MPEP exposure, which could also modify adenosine A2A receptors (A2AR) since functional cross-talk between them has been reported. Here we report that MPEP crosses placental barrier and reaches neonatal brain through maternal milk using LC-MS/MS methods. Therefore, we analyzed mGlu receptors, mainly mGlu5, and A2AR in both maternal and fetal brain after chronic maternal consumption of MPEP during gestation and/or lactation using radioligand binding, Western-blotting, real-time PCR and phospholipase C (PLC) activity assays. In maternal brain, chronic MPEP consumption caused a significant loss of mGlu, including mGlu5, and A2AR receptors level in plasma membrane. PLC activity assays showed that mGlu5 signaling pathway was desensitized. No variations on mRNA level coding A2AR, A1R and mGlu5 were found after MPEP treatments. In female neonatal brain, maternal consumption of MPEP caused a significant increase in mGlu, including mGlu5, and A2AR receptors level. Neither mGlu receptors nor A2AR were modified in male neonatal brain after maternal MPEP intake. Finally, neither molecular nor behavioral changes (anxiety- and depression-like behavior) were observed in 3-month-old female offspring. In summary, mGlu5 and A2AR are altered in both maternal and female neonatal brain after chronic maternal consumption of MPEP during gestation and/or lactation.

  1. Increased striatal adenosine A2A receptor levels is an early event in Parkinson's disease-related pathology and it is potentially regulated by miR-34b.

    PubMed

    Villar-Menéndez, Izaskun; Porta, Sílvia; Buira, Sandra P; Pereira-Veiga, Thais; Díaz-Sánchez, Sara; Albasanz, José Luis; Ferrer, Isidre; Martín, Mairena; Barrachina, Marta

    2014-09-01

    Adenosine A2A receptor (A2AR) is a G-protein coupled receptor that stimulates adenylyl cyclase activity. In the brain, A2ARs are found highly enriched in striatal GABAergic medium spiny neurons, related to the control of voluntary movement. Pharmacological modulation of A2ARs is particularly useful in Parkinson's disease (PD) due to their property of antagonizing dopamine D2 receptor activity. Increases in A2AR levels have been described in PD patients showing an important loss of dopaminergic denervation markers, but no data have been reported about A2AR levels in incidental PD brains. In the present report, we show that increased A2ARs protein levels were also detected in the putamen of incidental PD cases (Braak PD stages 1-2) with respect to age-matched controls. By contrast, A2ARs mRNA levels remained unchanged, suggesting that posttranslational mechanisms could be involved in the regulation of A2ARs. It has been described how miR-34b/c downregulation is an early event in PD cases. We found that miR-34b levels are also significantly reduced in the putamen of incidental PD cases and along disease progression. Given that 3'UTR of A2AR contains a predicted target site for miR-34b, the potential role of this miRNA in protein A2AR levels was assessed. In vitro studies revealed that endogenous A2AR protein levels increased when miR-34b function was blocked using a specific anti-miR-34b. Moreover, using a luciferase reporter assay with point mutations in a miR-34b predicted binding site within the 3'UTR region of A2AR mRNA abolished the effect of the miRNA using a miR-34b mimic. In addition, we showed a reduced percentage of DNA methylation in the 5'UTR region of ADORA2A in advanced PD cases. Overall, these findings reveal that increased A2AR protein levels occur in asymptomatic PD patients and provide new insights into the molecular mechanisms underlying A2AR expression levels along the progression of this neurodegenerative disease.

  2. Early synaptic deficits in the APP/PS1 mouse model of Alzheimer's disease involve neuronal adenosine A2A receptors

    PubMed Central

    Viana da Silva, Silvia; Haberl, Matthias Georg; Zhang, Pei; Bethge, Philipp; Lemos, Cristina; Gonçalves, Nélio; Gorlewicz, Adam; Malezieux, Meryl; Gonçalves, Francisco Q.; Grosjean, Noëlle; Blanchet, Christophe; Frick, Andreas; Nägerl, U Valentin; Cunha, Rodrigo A.; Mulle, Christophe

    2016-01-01

    Synaptic plasticity in the autoassociative network of recurrent connections among hippocampal CA3 pyramidal cells is thought to enable the storage of episodic memory. Impaired episodic memory is an early manifestation of cognitive deficits in Alzheimer's disease (AD). In the APP/PS1 mouse model of AD amyloidosis, we show that associative long-term synaptic potentiation (LTP) is abolished in CA3 pyramidal cells at an early stage. This is caused by activation of upregulated neuronal adenosine A2A receptors (A2AR) rather than by dysregulation of NMDAR signalling or altered dendritic spine morphology. Neutralization of A2AR by acute pharmacological inhibition, or downregulation driven by shRNA interference in a single postsynaptic neuron restore associative CA3 LTP. Accordingly, treatment with A2AR antagonists reverts one-trial memory deficits. These results provide mechanistic support to encourage testing the therapeutic efficacy of A2AR antagonists in early AD patients. PMID:27312972

  3. Sinomenine Protects against Lipopolysaccharide-Induced Acute Lung Injury in Mice via Adenosine A2A Receptor Signaling

    PubMed Central

    Li, Jun; Zhao, Li; He, Xie; Zeng, Yi-Jun; Dai, Shuang-Shuang

    2013-01-01

    Sinomenine (SIN) is a bioactive alkaloid extracted from the Chinese medicinal plant Sinomenium acutum, which is widely used in the clinical treatment of rheumatoid arthritis (RA). However, its role in acute lung injury (ALI) is unclear. In this study, we investigate the role of SIN in lipopolysaccharide (LPS)-induced ALI in mice. After ALI, lung water content and histological signs of pulmonary injury were attenuated, whereas the PaO2/FIO2 (P/F) ratios were elevated significantly in the mice pretreated with SIN. Additionally, SIN markedly inhibited inflammatory cytokine TNF-α and IL-1β expression levels as well as neutrophil infiltration in the lung tissues of the mice. Microarray analysis and real-time PCR showed that SIN treatment upregulated adenosine A2A receptor (A2AR) expression, and the protective effect of SIN was abolished in A2AR knockout mice. Further investigation in isolated mouse neutrophils confirmed the upregulation of A2AR by SIN and showed that A2AR-cAMP-PKA signaling was involved in the anti-inflammatory effect of SIN. Taken together, these findings demonstrate an A2AR-associated anti-inflammatory effect and the protective role of SIN in ALI, which suggests a potential novel approach to treat ALI. PMID:23555007

  4. Sesamin protects against renal ischemia reperfusion injury by promoting CD39-adenosine-A2AR signal pathway in mice.

    PubMed

    Li, Ke; Gong, Xia; Kuang, Ge; Jiang, Rong; Wan, Jingyuan; Wang, Bin

    2016-01-01

    Ischemia reperfusion injury (IRI) is a leading cause of acute kidney injury with high morbidity and mortality due to limited therapy. Here, we examine whether sesamin attenuates renal IRI in an animal model and explore the underlying mechanisms. Male mice were subjected to right renal ischemia for 30 min followed by reperfusion for 24 h with sesamin (100 mg/kg) during which the left kidney was removed. Renal damage and function were assessed subsequently. The results showed that sesamin reduced kidney ischemia reperfusion injury, as assessed by decreased serum creatinine (Scr) and Blood urea nitrogen (BUN), alleviated tubular damage and apoptosis. In addition, sesamin inhibited neutrophils infiltration and pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β production in IR-preformed kidney. Notably, sesamin promoted the expression of CD39, A2A adenosine receptor (A2AAR), and A2BAR mRNA and protein as well as adenosine production. Furthermore, CD39 inhibitor or A2AR antagonist abolished partly the protection of sesamin in kidney IRI. In conclusion, sesamin could effectively protect kidney from IRI by inhibiting inflammatory responses, which might be associated with promoting the adenosine-CD39-A2AR signaling pathway.

  5. Sesamin protects against renal ischemia reperfusion injury by promoting CD39-adenosine-A2AR signal pathway in mice

    PubMed Central

    Li, Ke; Gong, Xia; Kuang, Ge; Jiang, Rong; Wan, Jingyuan; Wang, Bin

    2016-01-01

    Ischemia reperfusion injury (IRI) is a leading cause of acute kidney injury with high morbidity and mortality due to limited therapy. Here, we examine whether sesamin attenuates renal IRI in an animal model and explore the underlying mechanisms. Male mice were subjected to right renal ischemia for 30 min followed by reperfusion for 24 h with sesamin (100 mg/kg) during which the left kidney was removed. Renal damage and function were assessed subsequently. The results showed that sesamin reduced kidney ischemia reperfusion injury, as assessed by decreased serum creatinine (Scr) and Blood urea nitrogen (BUN), alleviated tubular damage and apoptosis. In addition, sesamin inhibited neutrophils infiltration and pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β production in IR-preformed kidney. Notably, sesamin promoted the expression of CD39, A2A adenosine receptor (A2AAR), and A2BAR mRNA and protein as well as adenosine production. Furthermore, CD39 inhibitor or A2AR antagonist abolished partly the protection of sesamin in kidney IRI. In conclusion, sesamin could effectively protect kidney from IRI by inhibiting inflammatory responses, which might be associated with promoting the adenosine-CD39-A2AR signaling pathway. PMID:27347331

  6. Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

    DOE PAGES

    Naranjo, Andrea N.; McNeely, Patrick M.; Katsaras, John; ...

    2016-05-27

    The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, ormore » DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. Furthermore, the studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.« less

  7. Cocaine self-administration differentially affects allosteric A2A-D2 receptor-receptor interactions in the striatum. Relevance for cocaine use disorder.

    PubMed

    Pintsuk, Julia; Borroto-Escuela, Dasiel O; Pomierny, Bartosz; Wydra, Karolina; Zaniewska, Magdalena; Filip, Malgorzata; Fuxe, Kjell

    2016-05-01

    In the current study behavioral and biochemical experiments were performed to study changes in the allosteric A2AR-D2R interactions in the ventral and dorsal striatum after cocaine self-administration versus corresponding yoked saline control. By using ex vivo [(3)H]-raclopride/quinpirole competition experiments, the effects of the A2AR agonist CGS 21680 (100 nM) on the KiH and KiL values of the D2-like receptor (D2-likeR) were determined. One major result was a significant reduction in the D2-likeR agonist high affinity state observed with CGS 21680 after cocaine self-administration in the ventral striatum compared with the yoked saline group. The results therefore support the hypothesis that A2AR agonists can at least in part counteract the motivational actions of cocaine. This action is mediated via the D2-likeR by targeting the A2AR protomer of A2AR-D2-like R heteroreceptor complexes in the ventral striatum, which leads to the reduction of D2-likeR protomer recognition through the allosteric receptor-receptor interaction. In contrast, in the dorsal striatum the CGS 21680-induced antagonistic modulation in the D2-likeR agonist high affinity state was abolished after cocaine self-administration versus the yoked saline group probably due to a local dysfunction/disruption of the A2AR-D2-like R heteroreceptor complexes. Such a change in the dorsal striatum in cocaine self-administration can contribute to the development of either locomotor sensitization, habit-forming learning and/or the compulsive drug seeking by enhanced D2-likeR protomer signaling. Potential differences in the composition and stoichiometry of the A2AR-D2R heteroreceptor complexes, including differential recruitment of sigma 1 receptor, in the ventral and dorsal striatum may explain the differential regional changes observed in the A2A-D2-likeR interactions after cocaine self-administration.

  8. Histamine H3 Receptor Activation Counteracts Adenosine A2A Receptor-Mediated Enhancement of Depolarization-Evoked [3H]-GABA Release from Rat Globus Pallidus Synaptosomes

    PubMed Central

    2014-01-01

    High levels of histamine H3 receptors (H3Rs) are found in the globus pallidus (GP), a neuronal nucleus in the basal ganglia involved in the control of motor behavior. By using rat GP isolated nerve terminals (synaptosomes), we studied whether H3R activation modified the previously reported enhancing action of adenosine A2A receptor (A2AR) stimulation on depolarization-evoked [3H]-GABA release. At 3 and 10 nM, the A2AR agonist CGS-21680 enhanced [3H]-GABA release induced by high K+ (20 mM) and the effect of 3 nM CGS-21680 was prevented by the A2AR antagonist ZM-241385 (100 nM). The presence of presynaptic H3Rs was confirmed by the specific binding of N-α-[methyl-3H]-histamine to membranes from GP synaptosomes (maximum binding, Bmax, 1327 ± 79 fmol/mg protein; dissociation constant, Kd, 0.74 nM), which was inhibited by the H3R ligands immepip, clobenpropit, and A-331440 (inhibition constants, Ki, 0.28, 8.53, and 316 nM, respectively). Perfusion of synaptosomes with the H3R agonist immepip (100 nM) had no effect on K+-evoked [3H]-GABA release, but inhibited the stimulatory action of A2AR activation. In turn, the effect of immepip was blocked by the H3R antagonist clobenpropit, which had no significant effect of its own on K+-induced [3H]-GABA release. These data indicate that H3R activation selectively counteracts the facilitatory action of A2AR stimulation on GABA release from striato-pallidal projections. PMID:24884070

  9. Intrathecal injection of adenosine 2A receptor agonists reversed neuropathic allodynia through protein kinase (PK)A/PKC signaling.

    PubMed

    Loram, Lisa C; Taylor, Frederick R; Strand, Keith A; Harrison, Jacqueline A; Rzasalynn, Rachael; Sholar, Paige; Rieger, Jayson; Maier, Steven F; Watkins, Linda R

    2013-10-01

    A single intrathecal dose of adenosine 2A receptor (A2AR) agonist was previously reported to produce a multi-week reversal of allodynia in a chronic constriction injury (CCI) model of neuropathic pain. We aimed to determine if this long-term reversal was induced by A2AR agonism versus more generalized across adenosine receptor subtypes, and begin to explore the intracellular signaling cascades involved. In addition, we sought to identify whether the enduring effect could be extended to other models of neuropathic pain. We tested an A1R and A2BR agonist in CCI and found the same long duration effect with A2BR but not A1R agonism. An A2AR agonist (ATL313) produced a significant long-duration reversal of mechanical allodynia induced by long established CCI (administered 6 weeks after surgery), spinal nerve ligation and sciatic inflammatory neuropathy. To determine if ATL313 had a direct effect on glia, ATL313 was coadministered with lipopolysaccharide to neonatal microglia and astrocytes in vitro. ATL313 significantly attenuated TNFα production in both microglia and astrocytes but had no effect on LPS induced IL-10. Protein kinase C significantly reversed the ATL313 effects on TNFα in vitro in microglia and astrocytes, while a protein kinase A inhibitor only effected microglia. Both intrathecal PKA and PKC inhibitors significantly reversed the effect of the A2AR agonist on neuropathic allodynia. Therefore, A2AR agonists administered IT remain an exciting novel target for the treatment of neuropathic pain.

  10. Brain-derived neurotrophic factor mediates neuroprotection against Aβ-induced toxicity through a mechanism independent on adenosine 2A receptor activation.

    PubMed

    Jerónimo-Santos, André; Fonseca-Gomes, João; Guimarães, Diogo Andrade; Tanqueiro, Sara Ramalho; Ramalho, Rita Mira; Ribeiro, Joaquim Alexandre; Sebastião, Ana Maria; Diógenes, Maria José

    2015-01-01

    Brain-derived neurotrophic factor (BDNF) promotes neuronal survival through TrkB-FL activation. The activation of adenosine A2A receptors (A2AR) is essential for most of BDNF-mediated synaptic actions, such as synaptic plasticity, transmission and neurotransmitter release. We now aimed at evaluating the A2AR influence upon BDNF-mediated neuroprotection against Aβ25-35 toxicity in cultured neurons. Results showed that BDNF increases cell survival and reduces the caspase-3 and calpain activation induced by amyloid-β (Aβ) peptide, in a mechanism probably dependent on PLCγ pathway. This BDNF-mediated neuroprotection is not affected by A2AR activation or inhibition. Moreover neither activation nor inhibition of A2AR, per se, significantly influenced Aβ-induced neuronal death on calpain-mediated cleavage of TrkB induced by Aβ. In conclusion, these results suggest that, in opposition to the fast synaptic actions of BDNF, the neuroprotective actions of this neurotrophin against a strong Aβ insult do not require the activation of A2AR.

  11. Chronic and acute adenosine A2A receptor blockade prevents long-term episodic memory disruption caused by acute cannabinoid CB1 receptor activation.

    PubMed

    Mouro, Francisco M; Batalha, Vânia L; Ferreira, Diana G; Coelho, Joana E; Baqi, Younis; Müller, Christa E; Lopes, Luísa V; Ribeiro, Joaquim A; Sebastião, Ana M

    2017-05-01

    Cannabinoid-mediated memory impairment is a concern in cannabinoid-based therapies. Caffeine exacerbates cannabinoid CB1 receptor (CB1R)-induced memory deficits through an adenosine A1 receptor-mediated mechanism. We now evaluated how chronic or acute blockade of adenosine A2A receptors (A2ARs) affects long-term episodic memory deficits induced by a single injection of a selective CB1R agonist. Long-term episodic memory was assessed by the novel object recognition (NOR) test. Mice received an intraperitoneal (i.p.) injection of the CB1/CB2 receptor agonist WIN 55,212-2 (1 mg/kg) immediately after the NOR training, being tested for novelty recognition 24 h later. Anxiety levels were assessed by the Elevated Plus Maze test, immediately after the NOR. Mice were also tested for exploratory behaviour at the Open Field. For chronic A2AR blockade, KW-6002 (istradefylline) (3 mg/kg/day) was administered orally for 30 days; acute blockade of A2ARs was assessed by i.p. injection of SCH 58261 (1 mg/kg) administered either together with WIN 55,212-2 or only 30 min before the NOR test phase. The involvement of CB1Rs was assessed by using the CB1R antagonist, AM251 (3 mg/kg, i.p.). WIN 55,212-2 caused a disruption in NOR, an action absent in mice also receiving AM251, KW-6002 or SCH 58261 during the encoding/consolidation phase; SCH 58251 was ineffective if present during retrieval only. No effects were detected in the Elevated Plus maze or Open Field Test. The finding that CB1R-mediated memory disruption is prevented by antagonism of adenosine A2ARs, highlights a possibility to prevent cognitive side effects when therapeutic application of CB1R drugs is desired.

  12. Genetic blockade of adenosine A2A receptors induces cognitive impairments and anatomical changes related to psychotic symptoms in mice.

    PubMed

    Moscoso-Castro, Maria; Gracia-Rubio, Irene; Ciruela, Francisco; Valverde, Olga

    2016-07-01

    Schizophrenia is a chronic severe mental disorder with a presumed neurodevelopmental origin, and no effective treatment. Schizophrenia is a multifactorial disease with genetic, environmental and neurochemical etiology. The main theories on the pathophysiology of this disorder include alterations in dopaminergic and glutamatergic neurotransmission in limbic and cortical areas of the brain. Early hypotheses also suggested that nucleoside adenosine is a putative affected neurotransmitter system, and clinical evidence suggests that adenosine adjuvants improve treatment outcomes, especially in poorly responsive patients. Hence, it is important to elucidate the role of the neuromodulator adenosine in the pathophysiology of schizophrenia. A2A adenosine receptor (A2AR) subtypes are expressed in brain areas controlling motivational responses and cognition, including striatum, and in lower levels in hippocampus and cerebral cortex. The aim of this study was to characterize A2AR knockout (KO) mice with complete and specific inactivation of A2AR, as an animal model for schizophrenia. We performed behavioral, anatomical and neurochemical studies to assess psychotic-like symptoms in adult male and female KO and wild-type (WT) littermates. Our results show impairments in inhibitory responses and sensory gating in A2AR KO animals. Hyperlocomotion induced by d-amphetamine and MK-801 was reduced in KO animals when compared to WT littermates. Moreover, A2AR KO animals show motor disturbances, social and cognitive alterations. Finally, behavioral impairments were associated with enlargement of brain lateral ventricles and decreased BDNF levels in the hippocampus. These data highlight the role of adenosine in the pathophysiology of schizophrenia and provide new possibilities for the therapeutic management of schizophrenia.

  13. Effects of the adenosine A2A receptor antagonist SCH 58621 on cyclooxygenase-2 expression, glial activation, and brain-derived neurotrophic factor availability in a rat model of striatal neurodegeneration.

    PubMed

    Minghetti, Luisa; Greco, Anita; Potenza, Rosa Luisa; Pezzola, Antonella; Blum, David; Bantubungi, Kadiombo; Popoli, Patrizia

    2007-05-01

    Inhibition of adenosine A2A receptors (A2ARs) is neuroprotective in several experimental models of striatal diseases. However, the mechanisms elicited by A2AR blockade are only partially known, and critical aspects about the potential beneficial effects of A2AR antagonism in models of neurodegeneration still await elucidation. In the present study, we analyzed the influence of the selective A2AR antagonist SCH 58261 in a rat model of striatal excitotoxicity obtained by unilateral intrastriatal injection of quinolinic acid (QA). We found that SCH 58261 differently affected the expression of cyclooxygenase-2 (COX-2) induced by QA in cortex and striatum. The antagonist enhanced COX-2 expression in cortical neurons and prevented it in striatal microglia-like cells. Similarly, SCH 58261 differently regulated astrogliosis and microglial activation in the 2 brain regions. In addition, the A2AR antagonist prevented the QA-induced increase in striatal brain-derived neurotrophic factor levels. Because COX-2 activity has been linked to excitotoxic processes and because brain-derived neurotrophic factor depletion has been observed in mouse models as well as in patients with Huntington disease, we suggest that the final outcome of A2AR blockade (namely neuroprotection vs neurodegeneration) is likely to depend on the balance among its various and region-specific effects.

  14. Membrane cholesterol access into a G-protein-coupled receptor

    NASA Astrophysics Data System (ADS)

    Guixà-González, Ramon; Albasanz, José L.; Rodriguez-Espigares, Ismael; Pastor, Manuel; Sanz, Ferran; Martí-Solano, Maria; Manna, Moutusi; Martinez-Seara, Hector; Hildebrand, Peter W.; Martín, Mairena; Selent, Jana

    2017-02-01

    Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.

  15. Membrane cholesterol access into a G-protein-coupled receptor

    PubMed Central

    Guixà-González, Ramon; Albasanz, José L.; Rodriguez-Espigares, Ismael; Pastor, Manuel; Sanz, Ferran; Martí-Solano, Maria; Manna, Moutusi; Martinez-Seara, Hector; Hildebrand, Peter W.; Martín, Mairena; Selent, Jana

    2017-01-01

    Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs. PMID:28220900

  16. Activation of Adenosine 2A receptor inhibits neutrophil apoptosis in an autophagy-dependent manner in mice with systemic inflammatory response syndrome

    PubMed Central

    Liu, Yang-Wuyue; Yang, Ting; Zhao, Li; Ni, Zhenhong; Yang, Nan; He, Fengtian; Dai, Shuang-Shuang

    2016-01-01

    Systemic inflammatory response syndrome (SIRS) is an overwhelming whole body inflammation caused by infectious diseases or sterile insults. Neutrophils are the dominant participants during inflammation, and their survival and death determine the initiation as well as resolution of SIRS. Apoptosis and autophagy are two fundamental cellular processes that modulating cell fate, but their correlation and regulators in neutrophils under SIRS condition have not been elucidated. In this study, we demonstrated that high dose of LPS induced both apoptosis and autophagy of neutrophils in a mouse SIRS model and LPS-stimulated neutrophils in vitro. Moreover, we found that the adenosine 2A receptor (A2AR), a known anti-inflammatory G protein-coupled receptor (GPCR), could inhibit LPS-induced neutrophil apoptosis by suppressing the LPS-induced autophagy. Activation of A2AR suppressed LPS-induced autophagy by inhibiting the ROS-JNK pathway as well as promoting GPCR βϒ subunit–AKT signaling. The A2AR-inhibited autophagy suppressed apoptosis of neutrophils by blocking caspase8, caspase3 and PARP signaling. These findings not only increase our understandings of neutrophils’ fate and function in response to systemic inflammation, but also identify a novel anti-inflammatory role of A2AR in modulating neutrophils’ survival during inflammation. PMID:27647162

  17. Caffeine Reverts Memory But Not Mood Impairment in a Depression-Prone Mouse Strain with Up-Regulated Adenosine A2A Receptor in Hippocampal Glutamate Synapses.

    PubMed

    Machado, Nuno J; Simões, Ana Patrícia; Silva, Henrique B; Ardais, Ana Paula; Kaster, Manuella P; Garção, Pedro; Rodrigues, Diana I; Pochmann, Daniela; Santos, Ana Isabel; Araújo, Inês M; Porciúncula, Lisiane O; Tomé, Ângelo R; Köfalvi, Attila; Vaugeois, Jean-Marie; Agostinho, Paula; El Yacoubi, Malika; Cunha, Rodrigo A; Gomes, Catarina A

    2017-03-01

    Caffeine prophylactically prevents mood and memory impairments through adenosine A2A receptor (A2AR) antagonism. A2AR antagonists also therapeutically revert mood and memory impairments, but it is not known if caffeine is also therapeutically or only prophylactically effective. Since depression is accompanied by mood and memory alterations, we now explored if chronic (4 weeks) caffeine consumption (0.3 g/L) reverts mood and memory impairment in helpless mice (HM, 12 weeks old), a bred-based model of depression. HM displayed higher immobility in the tail suspension and forced swimming tests, greater anxiety in the elevated plus maze, and poorer memory performance (modified Y-maze and object recognition). HM also had reduced density of synaptic (synaptophysin, SNAP-25), namely, glutamatergic (vGluT1; -22 ± 7 %) and GABAergic (vGAT; -23 ± 8 %) markers in the hippocampus. HM displayed higher A2AR density (72 ± 6 %) in hippocampal synapses, an enhanced facilitation of hippocampal glutamate release by the A2AR agonist, CGS21680 (30 nM), and a larger LTP amplitude (54 ± 8 % vs. 21 ± 5 % in controls) that was restored to control levels (30 ± 10 %) by the A2AR antagonist, SCH58261 (50 nM). Notably, caffeine intake reverted memory deficits and reverted the loss of hippocampal synaptic markers but did not affect helpless or anxiety behavior. These results reinforce the validity of HM as an animal model of depression by showing that they also display reference memory deficits. Furthermore, caffeine intake selectively reverted memory but not mood deficits displayed by HM, which are associated with an increased density and functional impact of hippocampal A2AR controlling synaptic glutamatergic function.

  18. Cocaine-Induced Changes of Synaptic Transmission in the Striatum are Modulated by Adenosine A2A Receptors and Involve the Tyrosine Phosphatase STEP

    PubMed Central

    Chiodi, Valentina; Mallozzi, Cinzia; Ferrante, Antonella; Chen, Jiang F; Lombroso, Paul J; Di Stasi, Anna Maria Michela; Popoli, Patrizia; Domenici, Maria Rosaria

    2014-01-01

    The striatum is a brain area implicated in the pharmacological action of drugs of abuse. Adenosine A2A receptors (A2ARs) are highly expressed in the striatum and mediate, at least in part, cocaine-induced psychomotor effects in vivo. Here we studied the synaptic mechanisms implicated in the pharmacological action of cocaine in the striatum and investigated the influence of A2ARs. We found that synaptic transmission was depressed in corticostriatal slices after perfusion with cocaine (10 μM). This effect was reduced by the A2AR antagonist ZM241385 and almost abolished in striatal A2AR-knockout mice (mice lacking A2ARs in striatal neurons, stA2ARKO). The effect of cocaine on synaptic transmission was also prevented by the protein tyrosine phosphatases (PTPs) inhibitor sodium orthovanadate (Na3VO4). In synaptosomes prepared from striatal slices, we found that the activity of striatal-enriched protein tyrosine phosphatase (STEP) was upregulated by cocaine, prevented by ZM241385, and absent in synaptosomes from stA2ARKO. The role played by STEP in cocaine modulation of synaptic transmission was investigated in whole-cell voltage clamp recordings from medium spiny neurons of the striatum. We found that TAT-STEP, a peptide that renders STEP enzymatically inactive, prevented cocaine-induced reduction in AMPA- and NMDA-mediated excitatory post-synaptic currents, whereas the control peptide, TAT-myc, had no effect. These results demonstrate that striatal A2ARs modulate cocaine-induced synaptic depression in the striatum and highlight the potential role of PTPs and specifically STEP in the effects of cocaine. PMID:23989619

  19. Cocaine-induced changes of synaptic transmission in the striatum are modulated by adenosine A2A receptors and involve the tyrosine phosphatase STEP.

    PubMed

    Chiodi, Valentina; Mallozzi, Cinzia; Ferrante, Antonella; Chen, Jiang F; Lombroso, Paul J; Di Stasi, Anna Maria Michela; Popoli, Patrizia; Domenici, Maria Rosaria

    2014-02-01

    The striatum is a brain area implicated in the pharmacological action of drugs of abuse. Adenosine A2A receptors (A2ARs) are highly expressed in the striatum and mediate, at least in part, cocaine-induced psychomotor effects in vivo. Here we studied the synaptic mechanisms implicated in the pharmacological action of cocaine in the striatum and investigated the influence of A2ARs. We found that synaptic transmission was depressed in corticostriatal slices after perfusion with cocaine (10 μM). This effect was reduced by the A2AR antagonist ZM241385 and almost abolished in striatal A2AR-knockout mice (mice lacking A2ARs in striatal neurons, stA2ARKO). The effect of cocaine on synaptic transmission was also prevented by the protein tyrosine phosphatases (PTPs) inhibitor sodium orthovanadate (Na3VO4). In synaptosomes prepared from striatal slices, we found that the activity of striatal-enriched protein tyrosine phosphatase (STEP) was upregulated by cocaine, prevented by ZM241385, and absent in synaptosomes from stA2ARKO. The role played by STEP in cocaine modulation of synaptic transmission was investigated in whole-cell voltage clamp recordings from medium spiny neurons of the striatum. We found that TAT-STEP, a peptide that renders STEP enzymatically inactive, prevented cocaine-induced reduction in AMPA- and NMDA-mediated excitatory post-synaptic currents, whereas the control peptide, TAT-myc, had no effect. These results demonstrate that striatal A2ARs modulate cocaine-induced synaptic depression in the striatum and highlight the potential role of PTPs and specifically STEP in the effects of cocaine.

  20. Enduring Reversal Of Neuropathic Pain By A Single Intrathecal Injection Of Adenosine 2A Receptor Agonists: A Novel Therapy For Neuropathic Pain

    PubMed Central

    Loram, Lisa C; Harrison, Jacqueline A; Sloane, Evan M; Hutchinson, Mark R; Sholar, Paige; Taylor, Frederick R; Berkelhammer, Debra; Coats, Benjamen D; Poole, Stephen; Milligan, Erin D; Maier, Steven F; Rieger, Jayson; Watkins, Linda R

    2009-01-01

    Previous studies of peripheral immune cells have documented that activation of adenosine 2A receptors (A2AR) decrease pro-inflammatory cytokine release and increase release of the potent anti-inflammatory cytokine, interleukin-10 (IL-10). Given the growing literature supporting that glial proinflammatory cytokines importantly contribute to neuropathic pain, and that IL-10 can suppress such pain, we evaluated the effects of intrathecally (i.t.) administered A2AR agonists on neuropathic pain using the chronic constriction injury (CCI) model. A single i.t. injection of the A2AR agonists ATL313 or CGS21680, 10-14 d after CCI versus sham surgery, produced a long-duration reversal of mechanical allodynia and thermal hyperalgesia for at least 4 wk. Neither drug altered the nociceptive responses of sham-operated controls. An A2AR antagonist (ZM241385) co-administered i.t. with ATL313 abolished the action of ATL313 in rats with neuropathy-induced allodynia, but had no effect on allodynia in the absence of the A2AR agonist. ATL313 attenuated CCI-induced upregulation of spinal cord activation markers for microglia and astrocytes in the L4-L6 spinal cord segments both 1 wk and 4 wk after a single i.t. ATL313 administration. Neutralizing IL-10 antibodies administered i.t. transiently abolished the effect of ATL313 on neuropathic pain. In addition, IL-10 mRNA was significantly elevated in the CSF cells collected from the lumbar region. Activation of A2ARs following i.t. administration may be a novel, therapeutic approach for the treatment of neuropathic pain by increasing IL-10 in the immunocompetent cells of the CNS. PMID:19890011

  1. Caffeine consumption prevents memory impairment, neuronal damage, and adenosine A2A receptors upregulation in the hippocampus of a rat model of sporadic dementia.

    PubMed

    Espinosa, Janaína; Rocha, Andreia; Nunes, Fernanda; Costa, Marcelo S; Schein, Vanessa; Kazlauckas, Vanessa; Kalinine, Eduardo; Souza, Diogo O; Cunha, Rodrigo A; Porciúncula, Lisiane O

    2013-01-01

    Intracerebroventricular (icv) streptozotocin (STZ) administration induces pathological and behavioral alterations similar to those observed in Alzheimer's disease (AD) and is thus considered an experimental model of sporadic AD. Since caffeine (an adenosine receptor antagonist) and selective antagonists of adenosine A2A receptors modify the course of memory impairment in different amyloid-β-based experimental models of AD, we now tested the impact of caffeine on STZ-induced dementia and associated neurodegeneration in the hippocampus as well as on the expression and density of adenosine receptors. Adult male rats received a bilateral infusion of saline or STZ (3 mg/kg, icv), which triggered memory deficits after four weeks, as gauged by impaired object recognition memory. This was accompanied by a reduced NeuN immunoreactivity in the hippocampal CA1 region and an increased expression and density of adenosine A2A receptors (A2AR), but not A1R, in the hippocampus. Caffeine consumption (1 g/L in the drinking water starting 2 weeks before the STZ challenge) prevented the STZ-induced memory impairment and neurodegeneration as well as the upregulation of A2AR. These findings provide the first demonstration that caffeine prevents sporadic dementia and implicate the control of central A2AR as its likely mechanism of action.

  2. Epac2: a sulfonylurea receptor?

    PubMed

    Rehmann, Holger

    2012-02-01

    Sulfonylureas are widely used oral drugs in the treatment of diabetes mellitus. They function by the inhibition of ATP-sensitive K+ channels in pancreatic β-cells, which are thus considered the 'classical' sulfonylurea receptor. Next to the ATP-sensitive K+ channels, additional sulfonylurea-interacting proteins were identified, which might contribute to the physiological effects of this drug family. Most recently, Epac2 (exchange protein directly activated by cAMP 2) was added to the list of sulfonylurea receptors. However, this finding caused controversy in the literature. The critical discussion of the present paper comes to the conclusion that sulfonylureas are not able to activate Epac2 directly and are unlikely to bind to Epac2. Increased blood glucose levels after food intake result in the secretion of insulin from pancreatic β-cells. Glucose levels are detected 'indirectly' by β-cells: owing to increased glycolysis rates, the ratio of cellular ATP/ADP increases and causes the closure of ATP-sensitive K+ channels. In consequence, cells depolarize and voltage-dependent Ca2+ channels open to cause an increase in the cellular Ca2+ concentration. Finally, Ca2+ induces the fusion of insulin-containing granules with the plasma membrane. Sulfonylureas, such as tolbutamide, glibenclamide or acetohexamide, form a class of orally applicable drugs used in the treatment of non-insulin-dependent diabetes mellitus.

  3. The adenosine A2A receptor agonist CGS 21680 decreases ethanol self-administration in both non-dependent and dependent animals.

    PubMed

    Houchi, Hakim; Persyn, Wolfgang; Legastelois, Rémi; Naassila, Mickaël

    2013-09-01

    There is emerging evidence that the adenosinergic system might be involved in drug addiction and alcohol dependence. We have already demonstrated the involvement of A2A receptors (A2AR) in ethanol-related behaviours in mice. Here, we investigated whether the A2AR agonist CGS 21680 can reduce ethanol operant self-administration in both non-dependent and ethanol-dependent Wistar rats. To rule out a potential involvement of the A1R in the effects of CGS 21680, we also tested its effectiveness to reduce ethanol operant self-administration in both heterozygous and homozygous A1R knockout mice. Our results demonstrated that CGS 21680 (0.065, 0.095 and 0.125 mg/kg, i.p.) had a bimodal effect on 10% ethanol operant self-administration in non-dependent rats. The intermediate dose was also effective in reducing 2% sucrose self-administration. Interestingly, the intermediate dose reduced 10% ethanol self-administration in dependent animals more effectively (75% decrease) when compared with non-dependent animals (57% decrease). These results suggest that the A2AR are involved in CGS 21680 effects since the reduction of ethanol self-administration was not dependent upon the presence of A1R in mice. In conclusion, our findings demonstrated the effectiveness of the A2AR agonist CGS 21680 in a preclinical model of alcohol addiction and suggested that the adenosinergic pathway is a promising target to treat alcohol addiction.

  4. The stimulation of adenosine A2A receptors ameliorates the pathological phenotype of fibroblasts from Niemann-Pick type C patients.

    PubMed

    Visentin, Sergio; De Nuccio, Chiara; Bernardo, Antonietta; Pepponi, Rita; Ferrante, Antonella; Minghetti, Luisa; Popoli, Patrizia

    2013-09-25

    Niemann-Pick type C1 (NPC1) disease is a rare neurovisceral disorder characterized by intracellular accumulation of unesterified cholesterol, sphingolipids, and other lipids in the lysosomal compartment. A deregulation of lysosomal calcium has been identified as one of the earliest steps of the degenerative process. Since adenosine A2A receptors (A2ARs) control lysosome trafficking and pH, which closely regulates lysosomal calcium, we hypothesized a role for these receptors in NPC1. The aim of this study was to evaluate the effects of the A2AR agonist CGS21680 on human control and NPC1 fibroblasts. We show that CGS21680 raises lysosomal calcium levels and rescues mitochondrial functionality (mitochondrial inner membrane potential and expression of the complex IV of the mitochondrial respiratory chain), which is compromised in NPC1 cells. These effects are prevented by the selective blockade of A2ARs by the antagonist ZM241385. The effects of A2AR activation on lysosomal calcium are not mediated by the cAMP/PKA pathway but they appear to involve the phosphorylation of ERK1/2. Finally, CGS21680 reduces cholesterol accumulation (Filipin III staining), which is the main criterion currently used for identification of a compound or pathway that would be beneficial for NPC disease, and such an effect is prevented by the Ca(2+) chelator BAPTA-AM. Our findings strongly support the hypothesis that A2AR agonists may represent a therapeutic option for NPC1 and provide insights on their mechanisms of action.

  5. Ex vivo Perfusion with Adenosine A2A Receptor Agonist Enhances Rehabilitation of Murine Donor Lungs after Circulatory Death

    PubMed Central

    Stone, Mathew L.; Sharma, Ashish K.; Mas, Valeria. R.; Gehrau, Ricardo C.; Mulloy, Daniel P.; Zhao, Yunge; Lau, Christine L.; Kron, Irving L.; Laubach, Victor E.

    2015-01-01

    Background Ex vivo lung perfusion (EVLP) enables assessment and rehabilitation of marginal donor lungs prior to transplantation. We previously demonstrated that adenosine A2A receptor (A2AR) agonism attenuates lung ischemia-reperfusion injury. The current study utilizes a novel murine EVLP model to test the hypothesis that A2AR agonist enhances EVLP-mediated rehabilitation of donation after circulatory death (DCD) lungs. Methods Mice underwent euthanasia and 60 min warm ischemia, and lungs were flushed with Perfadex and underwent cold static preservation (CSP, 60 min). Three groups were studied: no EVLP (CSP), EVLP with Steen solution for 60 min (EVLP), and EVLP with Steen solution supplemented with ATL1223, a selective A2AR agonist (EVLP+ATL1223). Lung function, wet/dry weight, cytokines and neutrophil numbers were measured. Microarrays were performed using the Affymetrix GeneChip Mouse Genome 430A 2.0 Array. Results EVLP significantly improved lung function versus CSP, which was further, significantly improved by EVLP+ATL1223. Lung edema, cytokines and neutrophil counts were reduced after EVLP and further, significantly reduced after EVLP+ATL1223. Gene array analysis revealed differential expression of 1,594 genes after EVLP, which comprise canonical pathways involved in inflammation and innate immunity including IL-1, IL-8, IL-6 and IL-17 signaling. Several pathways were uniquely regulated by EVLP+ATL1223 including the downregulation of genes involved in IL-1 signaling such as ADCY9, ECSIT, IRAK1, MAPK12 and TOLLIP. Conclusion EVLP modulates pro-inflammatory genes and reduces pulmonary dysfunction, edema and inflammation in DCD lungs, which are further reduced by A2AR agonism. This murine EVLP model provides a novel platform to study rehabilitative mechanisms of DCD lungs. PMID:26262504

  6. Stimulation of adenosine A2A receptors reduces intracellular cholesterol accumulation and rescues mitochondrial abnormalities in human neural cell models of Niemann-Pick C1.

    PubMed

    Ferrante, A; De Nuccio, C; Pepponi, R; Visentin, S; Martire, A; Bernardo, A; Minghetti, L; Popoli, P

    2016-04-01

    Niemann Pick C 1 (NPC1) disease is an incurable, devastating lysosomal-lipid storage disorder characterized by hepatosplenomegaly, progressive neurological impairment and early death. Current treatments are very limited and the research of new therapeutic targets is thus mandatory. We recently showed that the stimulation of adenosine A2A receptors (A2ARs) rescues the abnormal phenotype of fibroblasts from NPC1 patients suggesting that A2AR agonists could represent a therapeutic option for this disease. However, since all NPC1 patients develop severe neurological symptoms which can be ascribed to the complex pathology occurring in both neurons and oligodendrocytes, in the present paper we tested the effects of the A2AR agonist CGS21680 in human neuronal and oligodendroglial NPC1 cell lines (i.e. neuroblastoma SH-SY5Y and oligodendroglial MO3.13 transiently transfected with NPC1 small interfering RNA). The down-regulation of the NPC1 protein effectively resulted in intracellular cholesterol accumulation and altered mitochondrial membrane potential. Both effects were significantly attenuated by CGS21680 (500 nM). The protective effects of CGS were prevented by the selective A2AR antagonist ZM241385 (500 nM). The involvement of calcium modulation was demonstrated by the ability of Bapta-AM (5-7 μM) in reverting the effect of CGS. The A2A-dependent activity was prevented by the PKA-inhibitor KT5720, thus showing the involvement of the cAMP/PKA signaling. These findings provide a clear in vitro proof of concept that A2AR agonists are promising potential drugs for NPC disease.

  7. FGF acts as a co-transmitter through Adenosine A2A receptor to regulate morphological and physiological synaptic plasticity

    PubMed Central

    Flajolet, Marc; Wang, Zhongfeng; Futter, Marie; Shen, Weixing; Nuangchamnong, Nina; Bendor, Jacob; Palaszewski, Iwona; Nairn, Angus C.; Surmeier, D. James; Greengard, Paul

    2009-01-01

    Summary Abnormalities of striatal function have been implicated in several major neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, and depression. Adenosine, by activation of A2A receptors, antagonizes dopamine signaling at D2 receptors and A2A receptor antagonists have been tested as therapeutic agents for Parkinson's disease. We report here a direct physical interaction between the G protein-coupled A2A receptor and the receptor tyrosine kinase FGF receptor. Concomitant activation of these two classes of receptors, but not individual activation of either one alone, causes a robust activation of the MAPK/ERK pathway, differentiation and neurite extension of PC12 cells, spine morphogenesis in primary neuronal cultures, and cortico-striatal plasticity induced by a novel A2AR/FGFR-dependent mechanism. The discovery of a direct physical interaction between the A2A and FGF receptors and the robust physiological consequences of this association shed light on the mechanism underlying FGF functions as a co-transmitter and open new avenues for therapeutic interventions. PMID:18953346

  8. Adenosine A2A Receptor Gene Knockout Prevents l-3,4-Dihydroxyphenylalanine-Induced Dyskinesia by Downregulation of Striatal GAD67 in 6-OHDA-Lesioned Parkinson’s Mice

    PubMed Central

    Yin, Su-bing; Zhang, Xiao-guang; Chen, Shuang; Yang, Wen-ting; Zheng, Xia-wei; Zheng, Guo-qing

    2017-01-01

    l-3,4-Dihydroxyphenylalanine (l-DOPA) remains the primary pharmacological agent for the symptomatic treatment of Parkinson’s disease (PD). However, the development of l-DOPA-induced dyskinesia (LID) limits the long-term use of l-DOPA for PD patients. Some data have reported that adenosine A2A receptor (A2AR) antagonists prevented LID in animal model of PD. However, the mechanism in which adenosine A2AR blockade alleviates the symptoms of LID has not been fully clarified. Here, we determined to knock out (KO) the gene of A2AR and explored the possible underlying mechanisms implicated in development of LID in a mouse model of PD. A2AR gene KO mice were unilaterally injected into the striatum with 6-hydroxydopamine (6-OHDA) in order to damage dopamine neurons on one side of the brain. 6-OHDA-lesioned mice were then injected once daily for 21 days with l-DOPA. Abnormal involuntary movements (AIMs) were evaluated on days 3, 8, 13, and 18 after l-DOPA administration, and real-time polymerase chain reaction and immunohistochemistry for glutamic acid decarboxylase (GAD) 65 and GAD67 were performed. We found that A2AR gene KO was effective in reducing AIM scores and accompanied with decrease of striatal GAD67, rather than GAD65. These results demonstrated that the possible mechanism involved in alleviation of AIM symptoms by A2AR gene KO might be through reducing the expression of striatal GAD67. PMID:28377741

  9. Effect of Caffeine Chronically Consumed During Pregnancy on Adenosine A1 and A2A Receptors Signaling in Both Maternal and Fetal Heart from Wistar Rats.

    PubMed

    Iglesias, Inmaculada; Albasanz, Jose Luis; Martín, Mairena

    2014-12-01

    Background: Caffeine is the most widely consumed psychoactive substance in the world, even during pregnancy. Its stimulatory effects are mainly due to antagonism of adenosine actions by blocking adenosine A1 and A2A receptors. Previous studies have shown that caffeine can cross the placenta and therefore modulate these receptors not only in the fetal brain but also in the heart. Methods: In the present work, the effect of caffeine chronically consumed during pregnancy on A1 and A2A receptors in Wistar rat heart, from both mothers and their fetuses, were studied using radioligand binding, Western-blotting, and adenylyl cyclase activity assays, as well as reverse transcription polymerase chain reaction. Results: Caffeine did not significantly alter A1R neither at protein nor at gene expression level in both the maternal and fetal heart. On the contrary, A2AR significantly decreased in the maternal heart, although mRNA was not affected. Gi and Gs proteins were also preserved. Finally, A1R-mediated inhibition of adenylyl cyclase activity did not change in the maternal heart, but A2AR mediated stimulation of this enzymatic activity significantly decreased according to the detected loss of this receptor. Conclusions: Opposite to the downregulation and desensitization of the A1R/AC pathway previously reported in the brain, these results show that this pathway is not affected in rat heart after caffeine exposure during pregnancy. In addition, A2AR is downregulated and desensitized in the maternal heart, suggesting a differential modulation of these receptor-mediated pathways by caffeine.

  10. Effect of Caffeine Chronically Consumed During Pregnancy on Adenosine A1 and A2A Receptors Signaling in Both Maternal and Fetal Heart from Wistar Rats

    PubMed Central

    Iglesias, Inmaculada; Albasanz, Jose Luis

    2014-01-01

    Background: Caffeine is the most widely consumed psychoactive substance in the world, even during pregnancy. Its stimulatory effects are mainly due to antagonism of adenosine actions by blocking adenosine A1 and A2A receptors. Previous studies have shown that caffeine can cross the placenta and therefore modulate these receptors not only in the fetal brain but also in the heart. Methods: In the present work, the effect of caffeine chronically consumed during pregnancy on A1 and A2A receptors in Wistar rat heart, from both mothers and their fetuses, were studied using radioligand binding, Western-blotting, and adenylyl cyclase activity assays, as well as reverse transcription polymerase chain reaction. Results: Caffeine did not significantly alter A1R neither at protein nor at gene expression level in both the maternal and fetal heart. On the contrary, A2AR significantly decreased in the maternal heart, although mRNA was not affected. Gi and Gs proteins were also preserved. Finally, A1R-mediated inhibition of adenylyl cyclase activity did not change in the maternal heart, but A2AR mediated stimulation of this enzymatic activity significantly decreased according to the detected loss of this receptor. Conclusions: Opposite to the downregulation and desensitization of the A1R/AC pathway previously reported in the brain, these results show that this pathway is not affected in rat heart after caffeine exposure during pregnancy. In addition, A2AR is downregulated and desensitized in the maternal heart, suggesting a differential modulation of these receptor-mediated pathways by caffeine. PMID:25538864

  11. Caffeine promotes anti-tumor immune response during tumor initiation: Involvement of the adenosine A2A receptor.

    PubMed

    Eini, Hadar; Frishman, Valeria; Yulzari, Robert; Kachko, Leonid; Lewis, Eli C; Chaimovitz, Cidio; Douvdevani, Amos

    2015-11-01

    Epidemiologic studies depict a negative correlation between caffeine consumption and incidence of tumors in humans. The main pharmacological effects of caffeine are mediated by antagonism of the adenosine receptor, A2AR. Here, we examine whether the targeting of A2AR by caffeine plays a role in anti-tumor immunity. In particular, the effects of caffeine are studied in wild-type and A2AR knockout (A2AR(-/-)) mice. Tumor induction was achieved using the carcinogen 3-methylcholanthrene (3-MCA). Alternatively, tumor cells, comprised of 3-MCA-induced transformed cells or B16 melanoma cells, were inoculated into animal footpads. Cytokine release was determined in a mixed lymphocyte tumor reaction (MLTR). According to our findings, caffeine-consuming mice (0.1% in water) developed tumors at a lower rate compared to water-consuming mice (14% vs. 53%, respectively, p=0.0286, n=15/group). Within the caffeine-consuming mice, tumor-free mice displayed signs of autoimmune alopecia and pronounced leukocyte recruitment intocarcinogen injection sites. Similarly, A2AR(-/-) mice exhibited reduced rates of 3-MCA-induced tumors. In tumor inoculation studies, caffeine treatment resulted in inhibition of tumor growth and elevation in proinflammatory cytokine release over water-consuming mice, as depicted by MLTR. Addition of the adenosine receptor agonist, NECA, to MLTR resulted in a sharp decrease in IFNγ levels; this was reversed by the highly selective A2AR antagonist, ZM241385. Thus, immune response modulation through either caffeine or genetic deletion of A2AR leads to a Th1 immune profile and suppression of carcinogen-induced tumorigenesis. Taken together, our data suggest that the use of pharmacologic A2AR antagonists may hold therapeutic potential in diminishing the rate of cancer development.

  12. Adenosine A2A receptors induced on iNKT and NK cells reduce pulmonary inflammation and injury in mice with sickle cell disease

    PubMed Central

    Wallace, Kori L.

    2010-01-01

    We showed previously that pulmonary function and arterial oxygen saturation in NY1DD mice with sickle cell disease (SCD) are improved by depletion of invariant natural killer T (iNKT) cells or blockade of their activation. Here we demonstrate that SCD causes a 9- and 6-fold induction of adenosine A2A receptor (A2AR) mRNA in mouse pulmonary iNKT and natural killer (NK) cells, respectively. Treating SCD mice with the A2AR agonist ATL146e produced a dose-dependent reversal of pulmonary dysfunction with maximal efficacy at 10 ng/kg/minute that peaked within 3 days and persisted throughout 7 days of continuous infusion. Crossing NY1DD mice with Rag1−/− mice reduced pulmonary injury that was restored by adoptive transfer of 106 purified iNKT cells. Reconstituted injury was reversed by ATL146e unless the adoptively transferred iNKT cells were pretreated with the A2AR alkylating antagonist, FSPTP (5-amino-7-[2-(4-fluorosulfonyl)phenylethyl]-2-(2-furyl)-pryazolo[4,3-ϵ]-1,2,4-triazolo[1,5-c]pyrimidine), which completely prevented pro-tection. In NY1DD mice exposed to hypoxia-reoxygenation, treatment with ATL146e at the start of reoxygenation prevented further lung injury. Together, these data indicate that activation of induced A2ARs on iNKT and NK cells in SCD mice is sufficient to improve baseline pulmonary function and prevent hypoxia-reoxygenation–induced exacerbation of pulmonary injury. A2A agonists have promise for treating diseases associated with iNKT or NK cell activation. PMID:20798237

  13. Understanding the Functional Plasticity in Neural Networks of the Basal Ganglia in Cocaine Use Disorder: A Role for Allosteric Receptor-Receptor Interactions in A2A-D2 Heteroreceptor Complexes

    PubMed Central

    Borroto-Escuela, Dasiel O.; Wydra, Karolina; Pintsuk, Julia; Narvaez, Manuel; Corrales, Fidel; Zaniewska, Magdalena; Agnati, Luigi F.; Franco, Rafael; Tanganelli, Sergio; Filip, Malgorzata

    2016-01-01

    Our hypothesis is that allosteric receptor-receptor interactions in homo- and heteroreceptor complexes may form the molecular basis of learning and memory. This principle is illustrated by showing how cocaine abuse can alter the adenosine A2AR-dopamine D2R heterocomplexes and their receptor-receptor interactions and hereby induce neural plasticity in the basal ganglia. Studies with A2AR ligands using cocaine self-administration procedures indicate that antagonistic allosteric A2AR-D2R heterocomplexes of the ventral striatopallidal GABA antireward pathway play a significant role in reducing cocaine induced reward, motivation, and cocaine seeking. Anticocaine actions of A2AR agonists can also be produced at A2AR homocomplexes in these antireward neurons, actions in which are independent of D2R signaling. At the A2AR-D2R heterocomplex, they are dependent on the strength of the antagonistic allosteric A2AR-D2R interaction and the number of A2AR-D2R and A2AR-D2R-sigma1R heterocomplexes present in the ventral striatopallidal GABA neurons. It involves a differential cocaine-induced increase in sigma1Rs in the ventral versus the dorsal striatum. In contrast, the allosteric brake on the D2R protomer signaling in the A2AR-D2R heterocomplex of the dorsal striatopallidal GABA neurons is lost upon cocaine self-administration. This is potentially due to differences in composition and allosteric plasticity of these complexes versus those in the ventral striatopallidal neurons. PMID:27872762

  14. Untangling dopamine-adenosine receptor-receptor assembly in experimental parkinsonism in rats

    PubMed Central

    Fernández-Dueñas, Víctor; Taura, Jaume J.; Cottet, Martin; Gómez-Soler, Maricel; López-Cano, Marc; Ledent, Catherine; Watanabe, Masahiko; Trinquet, Eric; Pin, Jean-Philippe; Luján, Rafael; Durroux, Thierry; Ciruela, Francisco

    2015-01-01

    Parkinson’s disease (PD) is a dopaminergic-related pathology in which functioning of the basal ganglia is altered. It has been postulated that a direct receptor-receptor interaction – i.e. of dopamine D2 receptor (D2R) with adenosine A2A receptor (A2AR) (forming D2R-A2AR oligomers) – finely regulates this brain area. Accordingly, elucidating whether the pathology prompts changes to these complexes could provide valuable information for the design of new PD therapies. Here, we first resolved a long-standing question concerning whether D2R-A2AR assembly occurs in native tissue: by means of different complementary experimental approaches (i.e. immunoelectron microscopy, proximity ligation assay and TR-FRET), we unambiguously identified native D2R-A2AR oligomers in rat striatum. Subsequently, we determined that, under pathological conditions (i.e. in a rat PD model), D2R-A2AR interaction was impaired. Collectively, these results provide definitive evidence for alteration of native D2R-A2AR oligomers in experimental parkinsonism, thus conferring the rationale for appropriate oligomer-based PD treatments. PMID:25398851

  15. Initial Evaluation of a Novel Adenosine A2A Receptor Ligand, (11)C-Preladenant, in Healthy Human Subjects.

    PubMed

    Sakata, Muneyuki; Ishibashi, Kenji; Imai, Masamichi; Wagatsuma, Kei; Ishii, Kenji; Zhou, Xiaoyun; de Vries, Erik F; Elsinga, Philip H; Ishiwata, Kiichi; Toyohara, Jun

    2017-03-09

    (11)C-Preladenant is a novel selective antagonist for mapping of cerebral adenosine A2A receptors (A2ARs) by positron emission tomography (PET). This is a first-in-human study to examine the safety, radiation dosimetry, and brain imaging of (11)C-preladenant in healthy human subjects. Methods: Dynamic (11)C-preladenant PET scans (90 min) were performed in 5 healthy male subjects. During the scan, arterial blood was sampled at various time intervals, and the fraction of the parent compound in plasma was determined. For anatomic coregistration, T1-weighted magnetic resonance imaging was performed. The total distribution volume (VT) was estimated using one- and two-tissue compartment models (1T and 2T, respectively). Distribution volume ratio (DVR) was calculated from VT of target and reference region, and obtained with a non-invasive Logan graphical reference tissue method (LGRM) (t* = 30 min). The applicability of a shortened protocol as an alternative to the 90 min PET scan was investigated. Tracer biodistribution and dosimetry were determined in 3 healthy male subjects, using serial whole-body PET scan acquired over 2 h post (11)C-preladenant injection. Results: There were no serious adverse events in any of the subjects throughout the study period. (11)C-Preladenat readily entered the brain, with a peak uptake in the putamen and head of the caudate nucleus 30-40 min after tracer injection. Other brain regions showed rapid clearance of radioactivity. The regional distribution of (11)C-preladenant was consistent with known A2AR densities in the brain. At pseudoequilibrium (reached at 40 min after injection), stable target-to-cerebellar cortex ratios of around 3.8-10.0 were obtained. The 2T fit better than the 1T in the low-density A2AR regions. In contrast, there were no significant differences between 1T and 2T in the high A2AR density regions. DVRs in putamen and head of the caudate nucleus were around 3.8-10.3 when estimated using a LGRM with cerebellum as the

  16. Molecular Determinants of CGS21680 Binding to the Human Adenosine A2A Receptor.

    PubMed

    Lebon, Guillaume; Edwards, Patricia C; Leslie, Andrew G W; Tate, Christopher G

    2015-06-01

    The adenosine A2A receptor (A(2A)R) plays a key role in transmembrane signaling mediated by the endogenous agonist adenosine. Here, we describe the crystal structure of human A2AR thermostabilized in an active-like conformation bound to the selective agonist 2-[p-(2-carboxyethyl)phenylethyl-amino]-5'-N-ethylcarboxamido adenosine (CGS21680) at a resolution of 2.6 Å. Comparison of A(2A)R structures bound to either CGS21680, 5'-N-ethylcarboxamido adenosine (NECA), UK432097 [6-(2,2-diphenylethylamino)-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-tetrahydrofuran-2-yl]-N-[2-[[1-(2-pyridyl)-4-piperidyl]carbamoylamino]ethyl]purine-2-carboxamide], or adenosine shows that the adenosine moiety of the ligands binds to the receptor in an identical fashion. However, an extension in CGS21680 compared with adenosine, the (2-carboxyethyl)phenylethylamino group, binds in an extended vestibule formed from transmembrane regions 2 and 7 (TM2 and TM7) and extracellular loops 2 and 3 (EL2 and EL3). The (2-carboxyethyl)phenylethylamino group makes van der Waals contacts with side chains of amino acid residues Glu169(EL2), His264(EL3), Leu267(7.32), and Ile274(7.39), and the amine group forms a hydrogen bond with the side chain of Ser67(2.65). Of these residues, only Ile274(7.39) is absolutely conserved across the human adenosine receptor subfamily. The major difference between the structures of A(2A)R bound to either adenosine or CGS21680 is that the binding pocket narrows at the extracellular surface when CGS21680 is bound, due to an inward tilt of TM2 in that region. This conformation is stabilized by hydrogen bonds formed by the side chain of Ser67(2.65) to CGS21680, either directly or via an ordered water molecule. Mutation of amino acid residues Ser67(2.65), Glu169(EL2), and His264(EL3), and analysis of receptor activation either in the presence or absence of ligands implicates this region in modulating the level of basal activity of A(2A)R.

  17. Cannabinoid 2 receptor- and beta Arrestin 2-dependent upregulation of serotonin 2A receptors.

    PubMed

    Franklin, J M; Vasiljevik, T; Prisinzano, T E; Carrasco, G A

    2013-07-01

    Recent evidence suggests that cannabinoid receptor agonists may regulate serotonin 2A (5-HT(2A)) receptor neurotransmission in the brain, although no molecular mechanism has been identified. Here, we present experimental evidence that sustained treatment with a non-selective cannabinoid agonist (CP55,940) or selective CB2 receptor agonists (JWH133 or GP1a) upregulate 5-HT(2A) receptors in a neuronal cell line. Furthermore, this cannabinoid receptor agonist-induced upregulation of 5-HT(2A) receptors was prevented in cells stably transfected with either CB2 or β-Arrestin 2 shRNA lentiviral particles. Additionally, inhibition of clathrin-mediated endocytosis also prevented the cannabinoid receptor-induced upregulation of 5-HT(2A) receptors. Our results indicate that cannabinoid agonists might upregulate 5-HT(2A) receptors by a mechanism that requires CB2 receptors and β-Arrestin 2 in cells that express both CB2 and 5-HT(2A) receptors. 5-HT(2A) receptors have been associated with several physiological functions and neuropsychiatric disorders such as stress response, anxiety and depression, and schizophrenia. Therefore, these results might provide a molecular mechanism by which activation of cannabinoid receptors might be relevant to some cognitive and mood disorders in humans.

  18. Dual blockade of the A1 and A2A adenosine receptor prevents amyloid beta toxicity in neuroblastoma cells exposed to aluminum chloride.

    PubMed

    Giunta, Salvatore; Andriolo, Violetta; Castorina, Alessandro

    2014-09-01

    In a previous work we have shown that exposure to aluminum (Al) chloride (AlCl3) enhanced the neurotoxicity of the amyloid beta(25-35) fragment (Abeta(25-35)) in neuroblastoma cells and affected the expression of Alzheimer's disease (AD)-related genes. Caffein, a compound endowed with beneficial effects against AD, exerts neuroprotection primarily through its antagonist activity on A2A adenosine receptors (A2AR), although it also inhibits A1Rs with similar potency. Still, studies on the specific involvement of these receptors in neuroprotection in a model of combined neurotoxicity (Abeta(25-35)+AlCl3) are missing. To address this issue, cultured SH-SY5Y cells exposed to Abeta(25-35)+AlCl3 were assessed for cell viability, morphology, intracellular ROS activity and expression of apoptosis-, stress- and AD-related proteins. To define the role of A1R and A2ARs, pretreatment with caffein, specific receptor antagonists (DPCPX or SCH58261) or siRNA-mediated gene knockdown were delivered. Results indicate that AlCl3 treatment exacerbated Abeta(25-35) toxicity, increased ROS production, lipid peroxidation, β-secretase-1 (BACE1) and amyloid precursor protein (APP). Interestingly, SCH58261 successfully prevented toxicity associated to Abeta(25-35) only, whereas pretreatment with both DPCPX and SCH58261 was required to fully avert Abeta(25-35)+AlCl3-induced damage, suggesting that A1Rs might also be critically involved in protection during combined toxicity. The effects of caffein were mimicked by both N-acetyl cysteine, an antioxidant, and desferrioxamine, likely acting through distinct mechanisms. Altogether, our data establish a novel protective function associated with A1R inhibition in the setting of combined Abeta(25-35)+AlCl3 neurotoxicity, and expand our current knowledge on the potential beneficial role of caffein to prevent AD progression in subjects environmentally exposed to aluminum.

  19. Agonist Derived Molecular Probes for A2A Adenosine Receptors

    PubMed Central

    Jacobson, Kenneth A.; Pannell, Lewis K.; Ji, Xiao-duo; Jarvis, Michael F.; Williams, Michael; Hutchison, Alan J.; Barrington, William W.; Stiles, Gary L.

    2011-01-01

    The adenosine agonist 2-(4-(2-carboxyethyl)phenylethylamino)-5′-N-ethylcarboxamidoadenosine (CGS21680) was recently reported to be selective for the A2A adenosine receptor subtype, which mediates its hypotensive action. To investigate structurelactivity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p-hydroxyphenylpropionic, 2-thiophenylacetic, and p-aminophenylacetic acids were prepared. The latter derivative (PAPA-APEC) was iodinated electrophilically using [125I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A, adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350-fold selective for A, receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl-1-piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta- and para-phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A2A receptors is relatively insensitive to distal structural changes at the 2-position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity labelling methodology. PMID:2561548

  20. Inosine, an Endogenous Purine Nucleoside, Suppresses Immune Responses and Protects Mice from Experimental Autoimmune Encephalomyelitis: a Role for A2A Adenosine Receptor.

    PubMed

    Junqueira, Stella Célio; Dos Santos Coelho, Igor; Lieberknecht, Vicente; Cunha, Mauricio Peña; Calixto, João B; Rodrigues, Ana Lúcia S; Santos, Adair Roberto Soares; Dutra, Rafael Cypriano

    2016-04-30

    Multiple sclerosis (MS) is a T cell autoimmune, inflammatory, and demyelinating disease of the central nervous system (CNS). Currently available therapies have partially effective actions and numerous side reactions. Inosine, an endogenous purine nucleoside, has immunomodulatory, neuroprotective, and analgesic properties. Herein, we evaluated the effect of inosine on the development and progression of experimental autoimmune encephalomyelitis (EAE), an experimental model of MS. Inosine (1 or 10 mg/kg, i.p.) was administrated twice a day for 40 days. Immunological and inflammatory responses were evaluated by behavioral, histological, immunohistochemical, ELISA, RT-PCR, and Western blotting analysis. The administration of inosine exerted neuroprotective effects against EAE by diminishing clinical signs, including thermal and mechanical hyperalgesia, as well as weight loss typical of the disease. These beneficial effects of inosine seem to be associated with the blockade of inflammatory cell entry into the CNS, especially lymphocytes, thus delaying the demyelinating process and astrocytes activation. In particular, up-regulation of IL-17 levels in the secondary lymphoid tissues, a result of EAE, was prevented by inosine treatment in EAE mice. Additionally, inosine consistently prevented A2AR up-regulation in the spinal cord, likely, through an ERK1-independent pathway. Altogether, these results allow us to propose that this endogenous purine might be a putative novel and helpful tool for the prevention of autoimmune and neurodegenerative diseases, such as MS. Thus, inosine could have considerable implications for future therapies of MS, and this study may represent the starting point for further investigation into the role of inosine and adenosinergic receptors in neuroinflammation processes. Graphical Abstract Preventive treatment with inosine inhibits the development and progression of EAE in C57Bl/6 mice. Furthermore, neuroinflammation and demyelinating processes

  1. Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors.

    PubMed

    Chiodi, Valentina; Ferrante, Antonella; Ferraro, Luca; Potenza, Rosa Luisa; Armida, Monica; Beggiato, Sarah; Pèzzola, Antonella; Bader, Michael; Fuxe, Kjell; Popoli, Patrizia; Domenici, Maria Rosaria

    2016-03-01

    Adenosine A2A receptors (A2 A Rs) and cannabinoid CB1 receptors (CB1 Rs) are highly expressed in the striatum, where they functionally interact and form A2A /CB1 heteroreceptor complexes. We investigated the effects of CB1 R stimulation in a transgenic rat strain over-expressing A2 A Rs under the control of the neural-specific enolase promoter (NSEA2A rats) and in age-matched wild-type (WT) animals. The effects of the CB1 R agonist WIN 55,212-2 (WIN) were significantly lower in NSEA2A rats than in WT animals, as demonstrated by i) electrophysiological recordings of synaptic transmission in corticostriatal slices; ii) the measurement of glutamate outflow from striatal synaptosomes and iii) in vivo experiments on locomotor activity. Moreover, while the effects of WIN were modulated by both A2 A R agonist (CGS 21680) and antagonists (ZM 241385, KW-6002 and SCH-442416) in WT animals, the A2 A R antagonists failed to influence WIN-mediated effects in NSEA2A rats. The present results demonstrate that in rats with genetic neuronal over-expression of A2 A Rs, the effects mediated by CB1 R activation in the striatum are significantly reduced, suggesting a change in the stoichiometry of A2A and CB1 receptors and providing a strategy to dissect the involvement of A2 A R forming or not forming heteromers in the modulation of striatal functions. These findings add additional evidence for the existence of an interaction between striatal A2 A Rs and CB1 Rs, playing a fundamental role in the regulation of striatal functions. We studied A2A -CB1 receptor interaction in transgenic rats over-expressing adenosine A2A receptors under the control of the neuron-specific enolase promoter (NSEA2A ). In these rats, we demonstrated a reduced effect of the CB1 receptor agonist WIN 55,212-2 in the modulation of corticostriatal synaptic transmission and locomotor activity, while CB1 receptor expression level did not change with respect to WT rats. A reduction in the expression of A2A -CB1

  2. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    SciTech Connect

    Sato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J.; Komai, Michio

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  3. BDNF prevents NMDA-induced toxicity in models of Huntington's disease: the effects are genotype specific and adenosine A2A receptor is involved.

    PubMed

    Martire, Alberto; Pepponi, Rita; Domenici, Maria Rosaria; Ferrante, Antonella; Chiodi, Valentina; Popoli, Patrizia

    2013-04-01

    NMDA receptor-mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain-derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A2 ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA-induced effects in HD models, and the possible involvement of A2ARs. In corticostriatal slices from wild-type mice and age-matched symptomatic R6/2 mice (a model of HD), NMDA application (75 μM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10 ng/mL) potentiated NMDA effects in wild-type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A2 AR blockade. The protective effect of BDNF against NMDA-induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A2 AR ligands in HD.

  4. Functional efficacy of adenosine A2A receptor agonists is positively correlated to their receptor residence time

    PubMed Central

    Guo, Dong; Mulder-Krieger, Thea; IJzerman, Adriaan P; Heitman, Laura H

    2012-01-01

    BACKGROUND AND PURPOSE The adenosine A2A receptor belongs to the superfamily of GPCRs and is a promising therapeutic target. Traditionally, the discovery of novel agents for the A2A receptor has been guided by their affinity for the receptor. This parameter is determined under equilibrium conditions, largely ignoring the kinetic aspects of the ligand-receptor interaction. The aim of this study was to assess the binding kinetics of A2A receptor agonists and explore a possible relationship with their functional efficacy. EXPERIMENTAL APPROACH We set up, validated and optimized a kinetic radioligand binding assay (a so-called competition association assay) at the A2A receptor from which the binding kinetics of unlabelled ligands were determined. Subsequently, functional efficacies of A2A receptor agonists were determined in two different assays: a novel label-free impedance-based assay and a more traditional cAMP determination. KEY RESULTS A simplified competition association assay yielded an accurate determination of the association and dissociation rates of unlabelled A2A receptor ligands at their receptor. A correlation was observed between the receptor residence time of A2A receptor agonists and their intrinsic efficacies in both functional assays. The affinity of A2A receptor agonists was not correlated to their functional efficacy. CONCLUSIONS AND IMPLICATIONS This study indicates that the molecular basis of different agonist efficacies at the A2A receptor lies within their different residence times at this receptor. PMID:22324512

  5. Adenosine 2A receptors in acute kidney injury.

    PubMed

    Vincent, I S; Okusa, M D

    2015-07-01

    Acute kidney injury (AKI) is an important clinical problem that may lead to death and for those who survive, the sequelae of AKI include loss of quality of life, chronic kidney disease and end-stage renal disease. The incidence of AKI continues to rise without clear successes in humans for the pharmacological prevention of AKI or treatment of established AKI. Dendritic cells and macrophages are critical early initiators of innate immunity in the kidney and orchestrate inflammation subsequent to ischaemia-reperfusion injury. These innate cells are the most abundant leucocytes present in the kidney, and they represent a heterogeneous population of cells that are capable of responding to cues from the microenvironment derived from pathogens or endogenous inflammatory mediators such as cytokines or anti-inflammatory mediators such as adenosine. Lymphocyte subsets such as natural killer T cells and Tregs also play roles in regulating ischaemic injury by promoting and suppressing inflammation respectively. Adenosine, produced in response to IR, is generally considered as a protective signalling molecule and elicits its physiological responses through four distinct adenosine receptors. However, its short half-life, lack of specificity and rapid metabolism limit the use of adenosine as a therapeutic agent. These adenosine receptors play various roles in regulating the activity of the aforementioned hematopoietic cells in elevated levels of adenosine such as during hypoxia. This review focuses on the importance of one receptor, the adenosine 2A subtype, in blocking inflammation associated with AKI.

  6. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A.

    PubMed

    Sato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J; Komai, Michio

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein-protein interactions with GR.

  7. Remodeling of striatal NMDA receptors by chronic A(2A) receptor blockade in Huntington's disease mice.

    PubMed

    Martire, Alberto; Ferrante, Antonella; Potenza, Rosa Luisa; Armida, Monica; Ferretti, Roberta; Pézzola, Antonella; Domenici, Maria Rosaria; Popoli, Patrizia

    2010-01-01

    Excitotoxicity plays a major role in the pathogenesis of Huntington disease (HD), a fatal neurodegenerative disorder. Adenosine A(2A) receptors (A(2A)Rs) modulate excitotoxicity and have been suggested to play a pathogenetic role in HD. The main aim of this study was to evaluate the effect of A(2A)R blockade on the expression and functions of NMDA receptors in the striatum of HD mice (R6/2). We found that 3 weeks' treatment with SCH 58261 (0.01 mg/kg/day i.p. from the 8th week of age) modified NR1 and NR2A/NR2B expression in the striatum of R6/2 (Western blotting) while had no effect on NMDA-induced toxicity in corticostriatal slices (electrophysiological experiments). In conclusion, in vivo A(2A)R blockade induced a remodeling of NMDA receptors in the striatum of HD mice. Even though the functional relevance of the above effect remains to be fully elucidated, these results add further evidence to the modulatory role of A(2A)Rs in HD.

  8. Guanosine may increase absence epileptic activity by means of A2A adenosine receptors in Wistar Albino Glaxo Rijswijk rats.

    PubMed

    Lakatos, Renáta Krisztina; Dobolyi, Árpád; Todorov, Mihail Ivilinov; Kékesi, Katalin A; Juhász, Gábor; Aleksza, Magdolna; Kovács, Zsolt

    2016-06-01

    The non-adenosine nucleoside guanosine (Guo) was demonstrated to decrease quinolinic acid(QA)-induced seizures, spontaneously emerged absence epileptic seizures and lipopolysaccharide(LPS)-evoked induction of absence epileptic seizures suggesting its antiepileptic potential. It was also described previously that intraperitoneal (i.p.) injection of 20 and 50mg/kg Guo decreased the number of spike-wave discharges (SWDs) in a well investigated model of human absence epilepsy, the Wistar Albino Glaxo Rijswijk (WAG/Rij) rats during 4th (20mg/kg Guo) and 3rd as well as 4th (50mg/kg Guo) measuring hours. Guanosine can potentially decrease SWD number by means of its putative receptors but absence epileptic activity changing effects of Guo by means of increased extracellular adenosine (Ado) cannot be excluded. An increase in the dose of i.p. injected Guo is limited by its low solubility in saline, therefore, we addressed in the present study whether higher doses of Guo, diluted in sodium hydroxide (NaOH) solution, have more potent antiepileptic effect in WAG/Rij rats. We confirmed that i.p. 50mg/kg Guo decreased but, surprisingly, i.p. 100mg/kg Guo enhanced the number of SWDs in WAG/Rij rats. Combined i.p. injection of a non-selective Ado receptor antagonist theophylline (5mg/kg) or a selective Ado A2A receptor (A2AR) antagonist SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) (1mg/kg) and a cyclooxygenase 1 and 2/COX-1 and COX-2 inhibitor indomethacin (10mg/kg) with 100mg/kg Guo decreased the SWD number compared to i.p. 100mg/kg Guo alone. The results suggest that i.p. 100mg/kg Guo can increase SWD number by means of the adenosinergic system.

  9. Involvement of cAMP-PKA pathway in adenosine A1 and A2A receptor-mediated regulation of acetaldehyde-induced activation of HSCs.

    PubMed

    Yang, Yaru; Wang, He; Lv, Xiongwen; Wang, Qi; Zhao, Han; Yang, Feng; Yang, Yan; Li, Jun

    2015-08-01

    The present study was undertaken to investigate the mechanism by which adenosine receptors (ARs)-mediated the cAMP/PKA/CREB signal pathway regulates the activation of acetaldehyde-induced hepatic stellate cells (HSCs). Primary HSCs were isolated from SD rats, cultured in vitro, and activated with different concentrations of acetaldehyde at different time points. Quantitative real-time PCR and Western blotting were used to quantify both protein and mRNA levels of the four AR (A1R, A2AR, A2BR, and A3R) in rat HSCs. Selective inhibitors of PDEs and the Gi/o protein pathway, general AR agonists, and AR subtype specific agents were used to study the AR signaling. The level of cAMP was measured by radio-immunoassay, and the expression of α-SMA, collagen type I and III, PKA and p-CREB were also detected by Western blotting. Acetaldehyde could significantly promote HSC proliferation, with a maximum stimulatory effect observed at 48 h after exposure to 200 μM acetaldehyde. All four AR subtypes could be present in rat HSCs, and the mRNA and protein expression levels for A2AR and A1R in much greater abundance than those for A2BR and A3R. The expression of A2AR and A1R was significantly increased in acetaldehyde-induced HSCs as compared with that of control group, whereas the expression of A2BR and A3R remained unaffected by the addition of acetaldehyde. Curiously, there is coupling of A2AR to the Gs-AC signaling, as well as coupling of A1R to the Gi/o-AC signaling pathway in acetaldehyde-induced HSCs. Both the A2AR and A1R antagonists could suppress the activation of HSC, although they have opposing effects on cAMP signal transduction. These results suggested that a combination of cAMP/PKA/CREB signals via A2AR and A1R likely mediate the activation of acetaldehyde-induced HSCs, and A1R coupled to the Gi/o-AC signaling pathway may be masked by the more predominant A2AR that coupled to the Gs-AC signaling pathway.

  10. Adenosine receptor neurobiology: overview.

    PubMed

    Chen, Jiang-Fan; Lee, Chien-fei; Chern, Yijuang

    2014-01-01

    Adenosine is a naturally occurring nucleoside that is distributed ubiquitously throughout the body as a metabolic intermediary. In the brain, adenosine functions as an important upstream neuromodulator of a broad spectrum of neurotransmitters, receptors, and signaling pathways. By acting through four G-protein-coupled receptors, adenosine contributes critically to homeostasis and neuromodulatory control of a variety of normal and abnormal brain functions, ranging from synaptic plasticity, to cognition, to sleep, to motor activity to neuroinflammation, and cell death. This review begun with an overview of the gene and genome structure and the expression pattern of adenosine receptors (ARs). We feature several new developments over the past decade in our understanding of AR functions in the brain, with special focus on the identification and characterization of canonical and noncanonical signaling pathways of ARs. We provide an update on functional insights from complementary genetic-knockout and pharmacological studies on the AR control of various brain functions. We also highlight several novel and recent developments of AR neurobiology, including (i) recent breakthrough in high resolution of three-dimension structure of adenosine A2A receptors (A2ARs) in several functional status, (ii) receptor-receptor heterodimerization, (iii) AR function in glial cells, and (iv) the druggability of AR. We concluded the review with the contention that these new developments extend and strengthen the support for A1 and A2ARs in brain as therapeutic targets for neurologic and psychiatric diseases.

  11. A2A-D2 receptor-receptor interaction modulates gliotransmitter release from striatal astrocyte processes.

    PubMed

    Cervetto, Chiara; Venturini, Arianna; Passalacqua, Mario; Guidolin, Diego; Genedani, Susanna; Fuxe, Kjell; Borroto-Esquela, Dasiel O; Cortelli, Pietro; Woods, Amina; Maura, Guido; Marcoli, Manuela; Agnati, Luigi F

    2017-01-01

    Evidence for striatal A2A-D2 heterodimers has led to a new perspective on molecular mechanisms involved in schizophrenia and Parkinson's disease. Despite the increasing recognition of astrocytes' participation in neuropsychiatric disease vulnerability, involvement of striatal astrocytes in A2A and D2 receptor signal transmission has never been explored. Here, we investigated the presence of D2 and A2A receptors in isolated astrocyte processes prepared from adult rat striatum by confocal imaging; the effects of receptor activation were measured on the 4-aminopyridine-evoked release of glutamate from the processes. Confocal analysis showed that A2A and D2 receptors were co-expressed on the same astrocyte processes. Evidence for A2A-D2 receptor-receptor interactions was obtained by measuring the release of the gliotransmitter glutamate: D2 receptors inhibited the glutamate release, while activation of A2A receptors, per se ineffective, abolished the effect of D2 receptor activation. The synthetic D2 peptide VLRRRRKRVN corresponding to the receptor region involved in electrostatic interaction underlying A2A-D2 heteromerization abolished the ability of the A2A receptor to antagonize the D2 receptor-mediated effect. Together, the findings are consistent with heteromerization of native striatal astrocytic A2A-D2 receptors that via allosteric receptor-receptor interactions could play a role in the control of striatal glutamatergic transmission. These new findings suggest possible new pathogenic mechanisms and/or therapeutic approaches to neuropsychiatric disorders.

  12. Potential therapeutic interest of adenosine A2A receptors in psychiatric disorders.

    PubMed

    Cunha, Rodrigo A; Ferré, Sergi; Vaugeois, Jean-Marie; Chen, Jiang-Fan

    2008-01-01

    The interest on targeting adenosine A(2A) receptors in the realm of psychiatric diseases first arose based on their tight physical and functional interaction with dopamine D(2) receptors. However, the role of central A(2A) receptors is now viewed as much broader than just controlling D(2) receptor function. Thus, there is currently a major interest in the ability of A(2A) receptors to control synaptic plasticity at glutamatergic synapses. This is due to a combined ability of A(2A) receptors to facilitate the release of glutamate and the activation of NMDA receptors. Therefore, A(2A) receptors are now conceived as a normalizing device promoting adequate adaptive responses in neuronal circuits, a role similar to that fulfilled, in essence, by dopamine. This makes A(2A) receptors particularly attractive targets to manage psychiatric disorders since adenosine may act as go-between glutamate and dopamine, two of the key players in mood processing. Furthermore, A(2A) receptors also control glia function and brain metabolic adaptation, two other emerging mechanisms to understand abnormal processing of mood, and A(2A) receptors are important players in controlling the demise of neurodegeneration, considered an amplificatory loop in psychiatric disorders. Current data only provide an indirect confirmation of this putative role of A(2A) receptors, based on the effects of caffeine (an antagonist of both A(1) and A(2A) receptors) in psychiatric disorders. However, the introduction of A(2A) receptors antagonists in clinics as anti-parkinsonian agents is hoped to bolster our knowledge on the role of A(2A) receptors in mood disorders in the near future.

  13. The importance of the adenosine A(2A) receptor-dopamine D(2) receptor interaction in drug addiction.

    PubMed

    Filip, M; Zaniewska, M; Frankowska, M; Wydra, K; Fuxe, K

    2012-01-01

    Drug addiction is a serious brain disorder with somatic, psychological, psychiatric, socio-economic and legal implications in the developed world. Illegal (e.g., psychostimulants, opioids, cannabinoids) and legal (alcohol, nicotine) drugs of abuse create a complex behavioral pattern composed of drug intake, withdrawal, seeking and relapse. One of the hallmarks of drugs that are abused by humans is that they have different mechanisms of action to increase dopamine (DA) neurotransmission within the mesolimbic circuitry of the brain and indirectly activate DA receptors. Among the DA receptors, D(2) receptors are linked to drug abuse and addiction because their function has been proven to be correlated with drug reinforcement and relapses. The recognition that D(2) receptors exist not only as homomers but also can form heteromers, such as with the adenosine (A)(2A) receptor, that are pharmacologically and functionally distinct from their constituent receptors, has significantly expanded the range of potential drug targets and provided new avenues for drug design in the search for novel drug addiction therapies. The aim of this review is to bring current focus on A(2A) receptors, their physiology and pharmacology in the central nervous system, and to discuss the therapeutic relevance of these receptors to drug addiction. We concentrate on the contribution of A(2A) receptors to the effects of different classes of drugs of abuse examined in preclinical behavioral experiments carried out with pharmacological and genetic tools. The consequences of chronic drug treatment on A(2A) receptor-assigned functions in preclinical studies are also presented. Finally, the neurochemical mechanism of the interaction between A(2A) receptors and drugs of abuse in the context of the heteromeric A(2A)-D(2) receptor complex is discussed. Taken together, a significant amount of experimental analyses provide evidence that targeting A(2A) receptors may offer innovative translational strategies

  14. 5-HT2A SEROTONIN RECEPTOR BIOLOGY: Interacting proteins, kinases and paradoxical regulation

    PubMed Central

    Roth, Bryan L

    2011-01-01

    5-hydroxytryptamine2A (5-HT2A) serotonin receptors are important pharmacological targets for a large number of central nervous system and peripheral serotonergic medications. In this review article I summarize work mainly from my lab regarding serotonin receptor anatomy, pharmacology, signaling and regulation. I highlight the role of serotonin receptor interacting proteins and the emerging paradigm of G-protein coupled receptor functional selectivity. PMID:21288474

  15. Adenosine A2A receptor antagonism and neuroprotection: mechanisms, lights, and shadows.

    PubMed

    Popoli, Patrizia; Minghetti, Luisa; Tebano, Maria Teresa; Pintor, Annita; Domenici, Maria Rosaria; Massotti, Marino

    2004-01-01

    Adenosine A2A receptor antagonists are regarded as potential neuroprotective drugs, although the mechanisms underlying their effects remain to be elucidated. In this review, quinolinic acid (QA)-induced striatal toxicity was used as a tool to investigate the mechanisms of the neuroprotective effects of A2A receptor antagonists. After having examined the effects of selective A2A receptor antagonists toward different mechanisms of QA toxicity, we conclude that (1) the effect elicited by A2A receptor blockade on QA-induced glutamate outflow may be one of the mechanisms of the neuroprotective activity of A2A receptor antagonists; (2) A2A receptor antagonists have a potentially worsening influence on QA-dependent NMDA receptor activation; and (3) the ability of A2A receptor antagonists to prevent QA-induced lipid peroxidation does not correlate with the neuroprotective effects. These results suggest that A2A receptor antagonists may have either potentially beneficial or detrimental influence in models of neurodegeneration that are mainly due to increased glutamate levels or enhanced sensitivity of NMDA receptors, respectively.

  16. Insights into the regulation of 5-HT2A serotonin receptors by scaffolding proteins and kinases.

    PubMed

    Allen, John A; Yadav, Prem N; Roth, Bryan L

    2008-11-01

    5-HT(2A) serotonin receptors are essential molecular targets for the actions of LSD-like hallucinogens and atypical antipsychotic drugs. 5-HT(2A) serotonin receptors also mediate a variety of physiological processes in peripheral and central nervous systems including platelet aggregation, smooth muscle contraction, and the modulation of mood and perception. Scaffolding proteins have emerged as important regulators of 5-HT(2A) receptors and our recent studies suggest multiple scaffolds exist for 5-HT(2A) receptors including PSD95, arrestin, and caveolin. In addition, a novel interaction has emerged between p90 ribosomal S6 kinase and 5-HT(2A) receptors which attenuates receptor signaling. This article reviews our recent studies and emphasizes the role of scaffolding proteins and kinases in the regulation of 5-HT(2A) trafficking, targeting and signaling.

  17. Inhibition of small HA fragment activity and stimulation of A2A adenosine receptor pathway limit apoptosis and reduce cartilage damage in experimental arthritis.

    PubMed

    Campo, Giuseppe M; Micali, Antonio; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Pisani, Antonina; Bruschetta, Antongiulio; Calatroni, Alberto; Puzzolo, Domenico; Campo, Salvatore

    2015-05-01

    Recent studies have found that the inactivation of small hyaluronan (HA) fragments originating from native HA during inflammation reduced the inflammatory response in models of experimental arthritis. The stimulation of adenosine receptors A2A reduced inflammation by inhibiting NF-kB activation. The combination of both treatments was significantly more effective than either of the individual treatments. The aim of this study was to further investigate the effects of a combined treatment using the HA inhibitor Pep-1 and a selective A2AR agonist (CV-1808) on the structure and ultrastructure of the articular cartilage and on apoptosis in a model of collagen-induced arthritis (CIA) in mice. Arthritic mice were treated with Pep-1 and/or CV-1808 intraperitoneally daily for 20 days. At day 35, the hind limbs were processed for light microscopy (hematoxylin/eosin and Safranin-O-Fast Green) and for transmission and scanning electron microscopy. CIA increased IL-6, caspase-3 and caspase-7 mRNA expression and the related protein levels in arthritic articular cartilage, and significantly increased concentrations of Bcl-2-associated X protein (Bax), while B cell-lymphoma-2 protein (Bcl-2) was markedly reduced. The combined Pep-1/CV-1808 treatment significantly reduced CIA injury, particularly at the highest doses, demonstrated by the presence of Safranin-O-positive cartilage, with a smooth surface and normal chondrocytes in the superficial, intermediate and deep zones. Morphological data and histological scoring were strongly supported by the reduction in inflammation and apoptotic markers. The results further support the role of HA degradation and A2A receptors in arthritis.

  18. Increased hypothalamic 5-HT2A receptor gene expression and effects of pharmacologic 5-HT2A receptor inactivation in obese A{sup y} mice

    SciTech Connect

    Nonogaki, Katsunori . E-mail: knonogaki-tky@umin.ac.jp; Nozue, Kana; Oka, Yoshitomo

    2006-12-29

    Serotonin (5-hydroxytryptamine; 5-HT) 2A receptors contribute to the effects of 5-HT on platelet aggregation and vascular smooth muscle cell proliferation, and are reportedly involved in decreases in plasma levels of adiponectin, an adipokine, in diabetic subjects. Here, we report that systemic administration of sarpogrelate, a 5-HT2A receptor antagonist, suppressed appetite and increased hypothalamic pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript, corticotropin releasing hormone, 5-HT2C, and 5-HT1B receptor gene expression. A{sup y} mice, which have ectopic expression of the agouti protein, significantly increased hypothalamic 5-HT2A receptor gene expression in association with obesity compared with wild-type mice matched for age. Systemic administration of sarpogrelate suppressed overfeeding, body weight gain, and hyperglycemia in obese A{sup y} mice, whereas it did not increase plasma adiponectin levels. These results suggest that obesity increases hypothalamic 5-HT2A receptor gene expression, and pharmacologic inactivation of 5-HT2A receptors inhibits overfeeding and obesity in A{sup y} mice, but did not increase plasma adiponectin levels.

  19. Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-Containing NMDA Receptors

    PubMed Central

    Li, Li-Jun; Hu, Rong; Lujan, Brendan; Chen, Juan; Zhang, Jian-Jian; Nakano, Yasuko; Cui, Tian-Yuan; Liao, Ming-Xia; Chen, Jin-Cao; Man, Heng-Ye; Feng, Hua; Wan, Qi

    2016-01-01

    NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs), but not GluN2B-containing NMDA receptors (GluN2BRs), to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation. PMID:27807405

  20. Activation, internalization, and recycling of the serotonin 2A receptor by dopamine

    PubMed Central

    Bhattacharyya, Samarjit; Raote, Ishier; Bhattacharya, Aditi; Miledi, Ricardo; Panicker, Mitradas M.

    2006-01-01

    Serotonergic and dopaminergic systems, and their functional interactions, have been implicated in the pathophysiology of various CNS disorders. Here, we use recombinant serotonin (5-HT) 2A (5-HT2A) receptors to further investigate direct interactions between dopamine and 5-HT receptors. Previous studies in Xenopus oocytes showed that dopamine, although not the cognate ligand for the 5-HT2A receptor, acts as a partial-efficacy agonist. At micromolar concentrations, dopamine also acts as a partial-efficacy agonist on 5-HT2A receptors in HEK293 cells. Like 5-HT, dopamine also induces receptor-internalization in these cells, although at significantly higher concentrations than 5-HT. Interestingly, if the receptors are first sensitized or “primed” by subthreshold concentrations of 5-HT, then dopamine-induced internalization occurs at concentrations ≈10-fold lower than when dopamine is used alone. Furthermore, unlike 5-HT-mediated internalization, dopamine-mediated receptor internalization, alone, or after sensitization by 5-HT, does not depend on PKC. Dopamine-internalized receptors recycle to the surface at rates similar to those of 5-HT-internalized receptors. Our results suggest a previously uncharacterized role for dopamine in the direct activation and internalization of 5-HT2A receptors that may have clinical relevance to the function of serotonergic systems in anxiety, depression, and schizophrenia and also to the treatment of these disorders. PMID:17005723

  1. Functions of 5-HT2A receptor and its antagonists in the cardiovascular system.

    PubMed

    Nagatomo, Takafumi; Rashid, Mamunur; Abul Muntasir, Habib; Komiyama, Tadazumi

    2004-10-01

    The serotonin (5-hydroxytryptamine, 5-HT) receptors have conventionally been divided into seven subfamilies, most of which have several subtypes. Among them, 5-HT(2A) receptor is associated with the contraction of vascular smooth muscle, platelet aggregation and thrombus formation and coronary artery spasms. Accordingly, selective 5-HT(2A) antagonists may have potential in the treatment of cardiovascular diseases. Sarpogrelate, a selective 5-HT(2A) antagonist, has been introduced clinically as a therapeutic agent for the treatment of ischemic diseases associated with thrombosis. Molecular modeling studies also suggest that sarpogrelate is a 5-HT(2A) selective antagonist and is likely to have pharmacological effects beneficial in the treatment of cardiovascular diseases. This review describes the above findings as well as the signaling linkages of the 5-HT(2A) receptors and the mode of agonist binding to 5-HT(2A) receptor using data derived from molecular modeling and site-directed mutagenesis.

  2. Lack of association between serotonin-2A receptor gene (HTR2A) polymorphisms and tardive dyskinesia in schizophrenia.

    PubMed

    Basile, V S; Ozdemir, V; Masellis, M; Meltzer, H Y; Lieberman, J A; Potkin, S G; Macciardi, F M; Petronis, A; Kennedy, J L

    2001-03-01

    Tardive dyskinesia (TD) is a disabling neurological side effect associated with long-term treatment with typical antipsychotics. Family studies and animal models lend evidence for hereditary predisposition to TD. The newer atypical antipsychotics pose a minimal risk for TD which is in part attributed to their ability to block the serotonin-2A (5-HT(2A)) receptor. 5-HT(2A) receptors were also identified in the basal ganglia; a brain region that plays a critical role in antipsychotic-induced movement disorders. We tested the significance of variation in the 5-HT(2A) receptor gene (HTR2A) in relation to the TD phenotype. Three polymorphisms in HTR2A, one silent (C102T), one that alters the amino acid sequence (his452tyr) and one in the promoter region (A-1437G) were investigated in 136 patients refractory or intolerant to treatment with typical antipsychotics and with a DSM-IIIR diagnosis of schizophrenia. We did not find any significant difference in allele, genotype or haplotype frequencies of polymorphisms in HTR2A among patients with or without TD (P > 0.05). Further analysis using the ANCOVA statistic with a continuous measure of the TD phenotype (Abnormal Involuntary Movement Scale (AIMS) score) found that the AIMS scores were not significantly influenced by HTR2A polymorphisms, despite controlling for potential confounders such as age, gender and ethnicity (P > 0.05). Theoretically, central serotonergic function can be subject to genetic control at various other mechanistic levels including the rate of serotonin synthesis (tryptophane hydroxylase gene), release, reuptake (serotonin transporter gene) and degradation (monoamine oxidase gene). Analyses of these other serotonergic genes are indicated. In summary, polymorphisms in HTR2A do not appear to influence the risk for TD. Further studies evaluating in tandem multiple candidate genes relevant for the serotonergic system are warranted to dissect the genetic basis of the complex TD phenotype.

  3. 5-HT2A receptor gene polymorphisms in Croatian subjects with autistic disorder.

    PubMed

    Hranilovic, Dubravka; Blazevic, Sofia; Babic, Marina; Smurinic, Maja; Bujas-Petkovic, Zorana; Jernej, Branimir

    2010-08-15

    Disturbances in the expression/function of the 5-HT2A receptor are implicated in autism. The association of the 5-HT2A receptor gene with autism was studied in the Croatian population. Distribution frequencies for alleles, genotypes and haplotypes of -1438 A/G and His452Tyr polymorphisms were compared in samples of 103 autistic and 214 control subjects. Significant overrepresentation of the G allele and the GG genotype of the -1438 A/G polymorphism was observed in group of autistic subjects, supporting the possible involvement of the 5-HT2A receptor in the development of autism.

  4. Positive association between a DNA sequence variant in the serotonin 2A receptor gene and schizophrenia

    SciTech Connect

    Inayama, Y.; Yoneda, H.; Sakai, T.

    1996-02-16

    Sixty-two patients with schizophrenia and 96 normal controls were investigated for genetic association with restriction fragment length polymorphisms (RFLPs) in the serotonin receptor genes. A positive association between the serotonin 2A receptor gene (HTR2A) and schizophrenia was found, but not between schizophrenia and the serotonin 1A receptor gene. The positive association we report here would suggest that the DNA region with susceptibility to schizophrenia lies in the HTR2A on the long arm of chromosome 13. 15 refs., 2 tabs.

  5. A critical evaluation of adenosine A2A receptors as potentially "druggable" targets in Huntington's disease.

    PubMed

    Popoli, Patrizia; Blum, David; Domenici, Maria Rosaria; Burnouf, Sylvie; Chern, Yijuang

    2008-01-01

    Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by the expansion of a polymorphic CAG trinucleotide repeat encoding a poly-glutamine tract within the Huntingtin protein. GABAergic enkephalin neurons of the basal ganglia, which show the highest levels of expression of adenosine A(2A) receptors, are the most vulnerable in HD. Such a selective neuronal vulnerability, which occurs despite ubiquitous expression of mutant and normal Huntingtin, has suggested that adenosine A(2A) receptors might play a pathogenetic role in HD. In agreement, changes in A(2A) receptor expression and signaling have been reported in various experimental models of HD. The interpretation of the functional significance of the aberrant A(2A) receptor phenotype in HD mice is however complicated by the conflicting data so far reported on the potential neuroprotective and neurodegenerative effects of these receptors in the brain, with some data suggesting a potential pathogenetic role and some other data suggesting activation of trophic or protective pathways in neurons. The same complex profile has emerged in experimental models of HD, in which both A(2A) receptor agonists and antagonists have shown beneficial effects. The main aim of this review is to critically evaluate whether adenosine A(2A) receptors may represent a suitable target to develop drugs against HD.

  6. GABAergic involvement in motor effects of an adenosine A(2A) receptor agonist in mice.

    PubMed

    Khisti, R T; Chopde, C T; Abraham, E

    2000-04-03

    Adenosine A(2A) agonists are known to induce catalepsy and inhibit dopamine mediated motor hyperactivity. An antagonistic interaction between adenosine A(2A) and dopamine D(2) receptors is known to regulate GABA-mediated neurotransmission in striatopallidal neurons. Stimulation of adenosine A(2A) and dopamine D(2) receptors has been shown to increase and inhibit GABA release respectively in pallidal GABAergic neurons. However, the role of GABAergic neurotransmission in the motor effects of adenosine A(2A) receptors is not yet known. Therefore in the present study the effect of GABAergic agents on adenosine A(2A) receptor agonist (NECA- or CGS 21680) induced catalepsy and inhibition of amphetamine elicited motor hyperactivity was examined. Pretreatment with GABA, the GABA(A) agonist muscimol or the GABA(B) agonist baclofen potentiated whereas the GABA(A) antagonist bicuculline attenuated NECA- or CGS 21680-induced catalepsy. However, the GABA(B) antagonists phaclophen and delta-aminovaleric acid had no effect. Administration of NECA or CGS 21680 not only reduced spontaneous locomotor activity but also antagonized amphetamine elicited motor hyperactivity. These effects of NECA and CGS 21680 were potentiated by GABA or muscimol and antagonized by bicuculline. These findings provide behavioral evidence for the role of GABA in the motor effects of adenosine A(2A) receptor agonists. Activation of adenosine A(2A) receptors increases GABA release which could reduce dopaminergic tone and induce catalepsy or inhibit amphetamine mediated motor hyperactivity.

  7. Adenosine A(2A) receptor gene (ADORA2A) variants may increase autistic symptoms and anxiety in autism spectrum disorder.

    PubMed

    Freitag, Christine M; Agelopoulos, Konstantin; Huy, Ellen; Rothermundt, Matthias; Krakowitzky, Petra; Meyer, Jobst; Deckert, Jürgen; von Gontard, Alexander; Hohoff, Christa

    2010-01-01

    Autism spectrum disorders (ASDs) are heterogeneous disorders presenting with increased rates of anxiety. The adenosine A(2A) receptor gene (ADORA2A) is associated with panic disorder and is located on chromosome 22q11.23. Its gene product, the adenosine A(2A) receptor, is strongly expressed in the caudate nucleus, which also is involved in ASD. As autistic symptoms are increased in individuals with 22q11.2 deletion syndrome, and large 22q11.2 deletions and duplications have been observed in ASD individuals, in this study, 98 individuals with ASD and 234 control individuals were genotyped for eight single-nucleotide polymorphisms in ADORA2A. Nominal association with the disorder was observed for rs2236624-CC, and phenotypic variability in ASD symptoms was influenced by rs3761422, rs5751876 and rs35320474. In addition, association of ADORA2A variants with anxiety was replicated for individuals with ASD. Findings point toward a possible mediating role of ADORA2A variants on phenotypic expression in ASD that need to be replicated in a larger sample.

  8. Lysergic acid diethylamide-induced Fos expression in rat brain: role of serotonin-2A receptors.

    PubMed

    Gresch, P J; Strickland, L V; Sanders-Bush, E

    2002-01-01

    Lysergic acid diethylamide (LSD) produces altered mood and hallucinations in humans and binds with high affinity to serotonin-2A (5-HT(2A)) receptors. Although LSD interacts with other receptors, the activation of 5-HT(2A) receptors is thought to mediate the hallucinogenic properties of LSD. The goal of this study was to identify the brain sites activated by LSD and to determine the influence of 5-HT(2A) receptors in this activation. Rats were pretreated with the 5-HT(2A) receptor antagonist MDL 100907 (0.3 mg/kg, i.p.) or vehicle 30 min prior to LSD (500 microg/kg, i.p.) administration and killed 3 h later. Brain tissue was examined for Fos protein expression by immunohistochemistry. LSD administration produced a five- to eight-fold increase in Fos-like immunoreactivity in medial prefrontal cortex, anterior cingulate cortex, and central nucleus of amygdala. However, in dorsal striatum and nucleus accumbens no increase in Fos-like immunoreactivity was observed. Pretreatment with MDL 100907 completely blocked LSD-induced Fos-like immunoreactivity in medial prefrontal cortex and anterior cingulate cortex, but only partially blocked LSD-induced Fos-like immunoreactivity in amygdala. Double-labeled immunohistochemistry revealed that LSD did not induce Fos-like immunoreactivity in cortical cells expressing 5-HT(2A) receptors, suggesting an indirect activation of cortical neurons. These results indicate that the LSD activation of medial prefrontal cortex and anterior cingulate cortex is mediated by 5-HT(2A) receptors, whereas in amygdala 5-HT(2A) receptor activation is a component of the response. These findings support the hypothesis that the medial prefrontal cortex, anterior cingulate cortex, and perhaps the amygdala, are important regions involved in the production of hallucinations.

  9. Adenosine A2A Receptors Modulate Acute Injury and Neuroinflammation in Brain Ischemia

    PubMed Central

    Pedata, Felicita; Pugliese, Anna Maria; Coppi, Elisabetta; Dettori, Ilaria; Maraula, Giovanna; Cellai, Lucrezia; Melani, Alessia

    2014-01-01

    The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A2A receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A2A receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by activation of resident immune cells, that is, microglia, and production or activation of inflammation mediators. Proinflammatory cytokines, which upregulate cell adhesion molecules, exert an important role in promoting recruitment of leukocytes that in turn promote expansion of the inflammatory response in ischemic tissue. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. A2A receptors present on central cells and on blood cells account for important effects depending on the time-related evolution of the pathological condition. Evidence suggests that A2A receptor antagonists provide early protection via centrally mediated control of excessive excitotoxicity, while A2A receptor agonists provide protracted protection by controlling massive blood cell infiltration in the hours and days after ischemia. Focus on inflammatory responses provides for adenosine A2A receptor agonists a wide therapeutic time-window of hours and even days after stroke. PMID:25165414

  10. Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory

    PubMed Central

    Orr, Anna G.; Hsiao, Edward C.; Wang, Max M.; Ho, Kaitlyn; Kim, Daniel H.; Wang, Xin; Guo, Weikun; Kang, Jing; Yu, Gui-Qiu; Adame, Anthony; Devidze, Nino; Dubal, Dena B.; Masliah, Eliezer; Conklin, Bruce R.; Mucke, Lennart

    2014-01-01

    Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimer’s disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice, and increased the levels of Arc/Arg3.1, an immediate-early gene required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Similar to humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss. PMID:25622143

  11. Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission.

    PubMed

    Martire, Alberto; Tebano, Maria Teresa; Chiodi, Valentina; Ferreira, Samira G; Cunha, Rodrigo A; Köfalvi, Attila; Popoli, Patrizia

    2011-01-01

    An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.

  12. Triggering neurotrophic factor actions through adenosine A2A receptor activation: implications for neuroprotection

    PubMed Central

    Sebastião, Ana M; Ribeiro, Joaquim A

    2009-01-01

    G protein coupled receptors and tropomyosin-related kinase (Trk) receptors have distinct structure and transducing mechanisms; therefore, cross-talk among them was unexpected. Evidence has, however, accumulated showing that tonic adenosine A2A receptor activity is a required step to allow synaptic actions of neurotrophic factors, namely upon synaptic transmission at both pre- and post-synaptic level as well as upon synaptic plasticity. An enhancement of A2A receptor tonus upon ageing may partially compensate the loss of TrkB receptors, rescuing to certain degree the facilitatory action of brain derived neurotrophic factor in aged animals, which might prove particularly relevant in the prevention of neurodegeneration upon ageing. A2A receptors also trigger synaptic actions of other neurotrophic factors, such as glial derived neurotrophic factor at dopaminergic striatal nerve endings. The growing evidence that tonic adenosine A2A receptor activity is a crucial step to allow actions of neurotrophic factors in neurones will be reviewed and discussed in the light of therapeutic strategies for neurodegenerative diseases. PMID:19508402

  13. 5-HT2A receptor activation is necessary for CO2-induced arousal

    PubMed Central

    Smith, Haleigh R.; MacAskill, Amanda; Richerson, George B.

    2015-01-01

    Hypercapnia-induced arousal from sleep is an important protective mechanism pertinent to a number of diseases. Most notably among these are the sudden infant death syndrome, obstructive sleep apnea and sudden unexpected death in epilepsy. Serotonin (5-HT) plays a significant role in hypercapnia-induced arousal. The mechanism of 5-HT's role in this protective response is unknown. Here we sought to identify the specific 5-HT receptor subtype(s) involved in this response. Wild-type mice were pretreated with antagonists against 5-HT receptor subtypes, as well as antagonists against adrenergic, cholinergic, histaminergic, dopaminergic, and orexinergic receptors before challenge with inspired CO2 or hypoxia. Antagonists of 5-HT2A receptors dose-dependently blocked CO2-induced arousal. The 5-HT2C receptor antagonist, RS-102221, and the 5-HT1A receptor agonist, 8-OH-DPAT, attenuated but did not completely block CO2-induced arousal. Blockade of non-5-HT receptors did not affect CO2-induced arousal. None of these drugs had any effect on hypoxia-induced arousal. 5-HT2 receptor agonists were given to mice in which 5-HT neurons had been genetically eliminated during embryonic life (Lmx1bf/f/p) and which are known to lack CO2-induced arousal. Application of agonists to 5-HT2A, but not 5-HT2C, receptors, dose-dependently restored CO2-induced arousal in these mice. These data identify the 5-HT2A receptor as an important mediator of CO2-induced arousal and suggest that, while 5-HT neurons can be independently activated to drive CO2-induced arousal, in the absence of 5-HT neurons and endogenous 5-HT, 5-HT receptor activation can act in a permissive fashion to facilitate CO2-induced arousal via another as yet unidentified chemosensor system. PMID:25925320

  14. A2A Adenosine Receptor Antagonism Enhances Synaptic and Motor Effects of Cocaine via CB1 Cannabinoid Receptor Activation

    PubMed Central

    Tozzi, Alessandro; de Iure, Antonio; Marsili, Valentina; Romano, Rosaria; Tantucci, Michela; Di Filippo, Massimiliano; Costa, Cinzia; Napolitano, Francesco; Mercuri, Nicola Biagio; Borsini, Franco; Giampà, Carmen; Fusco, Francesca Romana; Picconi, Barbara; Usiello, Alessandro; Calabresi, Paolo

    2012-01-01

    Background Cocaine increases the level of endogenous dopamine (DA) in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs) have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. Principal Findings Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. Conclusions The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine. PMID:22715379

  15. Photomodulation of G Protein-Coupled Adenosine Receptors by a Novel Light-Switchable Ligand

    PubMed Central

    2015-01-01

    The adenosinergic system operates through G protein-coupled adenosine receptors, which have become promising therapeutic targets for a wide range of pathological conditions. However, the ubiquity of adenosine receptors and the eventual lack of selectivity of adenosine-based drugs have frequently diminished their therapeutic potential. Accordingly, here we aimed to develop a new generation of light-switchable adenosine receptor ligands that change their intrinsic activity upon irradiation, thus allowing the spatiotemporal control of receptor functioning (i.e., receptor activation/inactivation dependent on location and timing). Therefore, we synthesized an orthosteric, photoisomerizable, and nonselective adenosine receptor agonist, nucleoside derivative MRS5543 containing an aryl diazo linkage on the N6 substituent, which in the dark (relaxed isomer) behaved as a full adenosine A3 receptor (A3R) and partial adenosine A2A receptor (A2AR) agonist. Conversely, upon photoisomerization with blue light (460 nm), it remained a full A3R agonist but became an A2AR antagonist. Interestingly, molecular modeling suggested that structural differences encountered within the third extracellular loop of each receptor could modulate the intrinsic, receptor subtype-dependent, activity. Overall, the development of adenosine receptor ligands with photoswitchable activity expands the pharmacological toolbox in support of research and possibly opens new pharmacotherapeutic opportunities. PMID:25248077

  16. Impaired Discrimination Learning in Mice Lacking the NMDA Receptor NR2A Subunit

    ERIC Educational Resources Information Center

    Brigman, Jonathan L.; Feyder, Michael; Saksida, Lisa M.; Bussey, Timothy J.; Mishina, Masayoshi; Holmes, Andrew

    2008-01-01

    N-Methyl-D-aspartate receptors (NMDARs) mediate certain forms of synaptic plasticity and learning. We used a touchscreen system to assess NR2A subunit knockout mice (KO) for (1) pairwise visual discrimination and reversal learning and (2) acquisition and extinction of an instrumental response requiring no pairwise discrimination. NR2A KO mice…

  17. Adenosine A2A receptors are necessary and sufficient to trigger memory impairment in adult mice

    PubMed Central

    Pagnussat, N; Almeida, A S; Marques, D M; Nunes, F; Chenet, G C; Botton, P H S; Mioranzza, S; Loss, C M; Cunha, R A; Porciúncula, L O

    2015-01-01

    Background and Purpose Caffeine (a non-selective adenosine receptor antagonist) prevents memory deficits in aging and Alzheimer’s disease, an effect mimicked by adenosine A2A receptor, but not A1 receptor, antagonists. Hence, we investigated the effects of adenosine receptor agonists and antagonists on memory performance and scopolamine-induced memory impairment in mice. Experimental Approach We determined whether A2A receptors are necessary for the emergence of memory impairments induced by scopolamine and whether A2A receptor activation triggers memory deficits in naïve mice, using three tests to assess short-term memory, namely the object recognition task, inhibitory avoidance and modified Y-maze. Key Results Scopolamine (1.0 mg·kg−1, i.p.) impaired short-term memory performance in all three tests and this scopolamine-induced amnesia was prevented by the A2A receptor antagonist (SCH 58261, 0.1–1.0 mg·kg−1, i.p.) and by the A1 receptor antagonist (DPCPX, 0.2–5.0 mg·kg−1, i.p.), except in the modified Y-maze where only SCH58261 was effective. Both antagonists were devoid of effects on memory or locomotion in naïve rats. Notably, the activation of A2A receptors with CGS 21680 (0.1–0.5 mg·kg−1, i.p.) before the training session was sufficient to trigger memory impairment in the three tests in naïve mice, and this effect was prevented by SCH 58261 (1.0 mg·kg−1, i.p.). Furthermore, i.c.v. administration of CGS 21680 (50 nmol) also impaired recognition memory in the object recognition task. Conclusions and Implications These results show that A2A receptors are necessary and sufficient to trigger memory impairment and further suggest that A1 receptors might also be selectively engaged to control the cholinergic-driven memory impairment. PMID:25939452

  18. Targeting Adenosine A2A Receptors in Parkinson’s Disease

    DTIC Science & Technology

    2006-11-01

    dysfunction in the rat hippocampus (collaboration with MA Lynch). Conclusion: These results prompt the hypothesis that the neuroprotection associated...stimuli and show reduced responses to morphine . The role of the A2A receptor in pain is less clear and we, and others, have shown the receptor is absent...cerebral cortex, striatum, hippocampus , olfactory bulb, and hypothalamus) but not in the cerebellum. (2), immunohistochemical analysis showed that

  19. Serotonin 2a Receptor and Serotonin 1a Receptor Interact Within the Medial Prefrontal Cortex During Recognition Memory in Mice

    PubMed Central

    Morici, Juan F.; Ciccia, Lucia; Malleret, Gaël; Gingrich, Jay A.; Bekinschtein, Pedro; Weisstaub, Noelia V.

    2015-01-01

    Episodic memory, can be defined as the memory for unique events. The serotonergic system one of the main neuromodulatory systems in the brain appears to play a role in it. The serotonin 2a receptor (5-HT2aR) one of the principal post-synaptic receptors for 5-HT in the brain, is involved in neuropsychiatric and neurological disorders associated with memory deficits. Recognition memory can be defined as the ability to recognize if a particular event or item was previously encountered and is thus considered, under certain conditions, a form of episodic memory. As human data suggest that a constitutively decrease of 5-HT2A signaling might affect episodic memory performance we decided to compare the performance of mice with disrupted 5-HT2aR signaling (htr2a−/−) with wild type (htr2a+/+) littermates in different recognition memory and working memory tasks that differed in the level of proactive interference. We found that ablation of 5-HT2aR signaling throughout development produces a deficit in tasks that cannot be solved by single item strategy suggesting that 5-HT2aR signaling is involved in interference resolution. We also found that in the absence of 5-HT2aR signaling serotonin has a deleterious effect on recognition memory retrieval through the activation of 5-HT1aR in the medial prefrontal cortex. PMID:26779016

  20. The A2a adenosine receptor modulates the reinforcement efficacy and neurotoxicity of MDMA.

    PubMed

    Ruiz-Medina, Jessica; Ledent, Catherine; Carretón, Olga; Valverde, Olga

    2011-04-01

    Adenosine is an endogenous purine nucleoside that plays a neuromodulatory role in the central nervous system. A2a adenosine receptors have been involved in reward-related processes, inflammatory phenomena and neurotoxicity reactions. In the present study, we investigated the role of A2a adenosine receptors on the acute pharmacological effects, reinforcement and neuroinflammation induced by MDMA administration. First, the acute effects of MDMA on body temperature, locomotor activity and anxiety-like responses were measured in A2a knockout mice and wild-type littermates. Second, MDMA reinforcing properties were evaluated using the intravenous self-administration paradigm. Finally, we assessed striatal astrogliosis and microgliosis as markers of MDMA neurotoxicity. Our results showed that acute MDMA produced a biphasic effect on body temperature and increased locomotor activity and anxiogenic-like responses in both genotypes. However, MDMA reinforcing properties were dramatically affected by the lack of A2a adenosine receptors. Thus, wild-type mice maintained MDMA self-administration under a fixed ratio 1 reinforcement schedule, whereas the operant response appeared completely abolished in A2a knockout mice. In addition, the MDMA neurotoxic regime produced an enhanced inflammatory response in striatum of wild-type mice, revealed by a significant increase in glial expression, whereas such activation was attenuated in mutant mice. This is the first report indicating that A2a adenosine receptors play a key role in reinforcement and neuroinflammation induced by the widely used psychostimulant.

  1. Mechanisms of the adenosine A2A receptor-induced sensitization of esophageal C fibers.

    PubMed

    Brozmanova, M; Mazurova, L; Ru, F; Tatar, M; Hu, Y; Yu, S; Kollarik, M

    2016-02-01

    Clinical studies indicate that adenosine contributes to esophageal mechanical hypersensitivity in some patients with pain originating in the esophagus. We have previously reported that the esophageal vagal nodose C fibers express the adenosine A2A receptor. Here we addressed the hypothesis that stimulation of the adenosine A2A receptor induces mechanical sensitization of esophageal C fibers by a mechanism involving transient receptor potential A1 (TRPA1). Extracellular single fiber recordings of activity originating in C-fiber terminals were made in the ex vivo vagally innervated guinea pig esophagus. The adenosine A2A receptor-selective agonist CGS21680 induced robust, reversible sensitization of the response to esophageal distention (10-60 mmHg) in a concentration-dependent fashion (1-100 nM). At the half-maximally effective concentration (EC50: ≈3 nM), CGS21680 induced an approximately twofold increase in the mechanical response without causing an overt activation. This sensitization was abolished by the selective A2A antagonist SCH58261. The adenylyl cyclase activator forskolin mimicked while the nonselective protein kinase inhibitor H89 inhibited mechanical sensitization by CGS21680. CGS21680 did not enhance the response to the purinergic P2X receptor agonist α,β-methylene-ATP, indicating that CGS21680 does not nonspecifically sensitize to all stimuli. Mechanical sensitization by CGS21680 was abolished by pretreatment with two structurally different TRPA1 antagonists AP18 and HC030031. Single cell RT-PCR and whole cell patch-clamp studies in isolated esophagus-specific nodose neurons revealed the expression of TRPA1 in A2A-positive C-fiber neurons and demonstrated that CGS21682 potentiated TRPA1 currents evoked by allylisothiocyanate. We conclude that stimulation of the adenosine A2A receptor induces mechanical sensitization of nodose C fibers by a mechanism sensitive to TRPA1 antagonists indicating the involvement of TRPA1.

  2. Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum.

    PubMed

    Tebano, Maria Teresa; Martire, Alberto; Chiodi, Valentina; Pepponi, Rita; Ferrante, Antonella; Domenici, Maria Rosaria; Frank, Claudio; Chen, Jiang-Fan; Ledent, Catherine; Popoli, Patrizia

    2009-09-01

    Adenosine A(2A), cannabinoid CB(1) and metabotropic glutamate 5 (mGlu(5)) receptors are all highly expressed in the striatum. The aim of the present work was to investigate whether, and by which mechanisms, the above receptors interact in the regulation of striatal synaptic transmission. By extracellular field potentials (FPs) recordings in corticostriatal slices, we demonstrated that the ability of the selective type 1 cannabinoid receptor (CB(1)R) agonist WIN55,212-2 to depress synaptic transmission was prevented by the pharmacological blockade or the genetic inactivation of A(2A)Rs. Such a permissive effect of A(2A)Rs towards CB(1)Rs does not seem to occur pre-synaptically as the ability of WIN55,212-2 to increase the R2/R1 ratio under a protocol of paired-pulse stimulation was not modified by ZM241385. Furthermore, the effects of WIN55,212-2 were reduced in slices from mice lacking post-synaptic striatal A(2A)Rs. The selective mGlu(5)R agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) potentiated the synaptic effects of WIN55,212-2, and such a potentiation was abolished by A(2A)R blockade. Unlike the synaptic effects, the ability of WIN55,212-2 to prevent NMDA-induced toxicity was not influenced by ZM241385. Altogether, these results show that the state of activation of A(2A)Rs regulates the synaptic effects of CB(1)Rs and that A(2A)Rs may control CB(1) effects also indirectly, namely through mGlu(5)Rs.

  3. Deletion of the G2A receptor fails to attenuate experimental autoimmune encephalomyelitis

    PubMed Central

    Osmers, Inga; Smith, Sherry S.; Parks, Brian W.; Yu, Shaohua; Srivastava, Roshni; Wohler, Jillian E.; Barnum, Scott R.; Kabarowski, Janusz H.S.

    2009-01-01

    Lysophosphatidylcholine (LPC) is a chemotactic lysolipid produced during inflammation by the hydrolytic action of phospholipase A2 enzymes. LPC stimulates chemotaxis of T cells in vitro through activation of the G protein-coupled receptor, G2A. This has led to the proposition that G2A contributes to the recruitment of T cells to sites of inflammation and thus promotes chronic inflammatory autoimmune diseases associated with the generation and subsequent tissue infiltration of auto-antigen-specific effector T cells. However, one study suggests that G2A may negatively regulate T cell proliferative responses to antigen receptor engagement and thereby attenuates autoimmunity by reducing the generation of autoreactive T cells. To address the relative contribution of these G2A-mediated effects to the pathophysiology of T cell-mediated autoimmune disease, we examined the impact of G2A inactivation on the onset and severity of murine experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Wild type (G2A+/+) and G2A-deficient (G2A-/-) C57BL/6J mice exhibited a similar incidence and onset of disease following immunization with MOG35-55 peptide. Disease severity was only moderately reduced in G2A-/- mice. Similar numbers of MOG35-55 specific T cells were generated in secondary lymphoid organs of MOG35-55-immunized G2A+/+ and G2A-/- mice. Comparable numbers of T cells were detected in spinal cords of G2A+/+ and G2A-/- mice. We conclude that the proposed anti-proliferative and chemotactic functions of G2A are not manifested in vivo and therefore therapeutic targeting of G2A is unlikely to be beneficial in the treatment of MS. PMID:19135725

  4. Expression of α(1)-adrenergic receptors in rat prefrontal cortex: cellular co-localization with 5-HT(2A) receptors.

    PubMed

    Santana, Noemí; Mengod, Guadalupe; Artigas, Francesc

    2013-06-01

    The prefrontal cortex (PFC) is involved in behavioural control and cognitive processes that are altered in schizophrenia. The brainstem monoaminergic systems control PFC function, yet the cells/networks involved are not fully known. Serotonin (5-HT) and norepinephrine (NE) increase PFC neuronal activity through the activation of α(1)-adrenergic receptors (α(1)ARs) and 5-HT(2A) receptors (5-HT(2A)Rs), respectively. Neurochemical and behavioural interactions between these receptors have been reported. Further, classical and atypical antipsychotic drugs share nm in vitro affinity for α(1)ARs while having preferential affinity for D(2) and 5-HT(2A)Rs, respectively. Using double in situ hybridization we examined the cellular expression of α(1)ARs in pyramidal (vGluT1-positive) and GABAergic (GAD(65/67)-positive) neurons in rat PFC and their co-localization with 5-HT(2A)Rs. α(1)ARs are expressed by a high proportion of pyramidal (59-85%) and GABAergic (52-79%) neurons. The expression in pyramidal neurons exhibited a dorsoventral gradient, with a lower percentage of α(1)AR-positive neurons in infralimbic cortex compared to anterior cingulate and prelimbic cortex. The expression of α(1A), α(1B) and α(1D) adrenergic receptors was segregated in different layers and subdivisions. In all them there is a high co-expression with 5-HT(2A)Rs (∼80%). These observations indicate that NE controls the activity of most PFC pyramidal neurons via α(1)ARs, either directly or indirectly, via GABAergic interneurons. Antipsychotic drugs can thus modulate the activity of PFC via α(1)AR blockade. The high co-expression with 5-HT(2A)Rs indicates a convergence of excitatory serotonergic and noradrenergic inputs onto the same neuronal populations. Moreover, atypical antipsychotics may exert a more powerful control of PFC function through the simultaneous blockade of α(1)ARs and 5-HT(2A)Rs.

  5. 5-HT2A receptors are involved in cognitive but not antidepressant effects of fluoxetine.

    PubMed

    Castañé, Anna; Kargieman, Lucila; Celada, Pau; Bortolozzi, Analía; Artigas, Francesc

    2015-08-01

    The prefrontal cortex (PFC) plays a crucial role in cognitive and affective functions. It contains a rich serotonergic (serotonin, 5-HT) innervation and a high density of 5-HT receptors. Endogenous 5-HT exerts robust actions on the activity of pyramidal neurons in medial PFC (mPFC) via excitatory 5-HT2A and inhibitory 5-HT1A receptors, suggesting the involvement of 5-HT neurotransmission in cortical functions. However, the underlying mechanisms must be elucidated. Here we examine the role of 5-HT2A receptors in the processing of emotional and cognitive signals evoked by increasing the 5-HT tone after acute blockade of the 5-HT transporter. Fluoxetine (5-20mg/kg i.p.) dose-dependently reduced the immobility time in the tail-suspension test in wild-type (WT) and 5-HT2Aknockout (KO2A) mice, with non-significant differences between genotypes. Fluoxetine (10mg/kg i.p.) significantly impaired mice performance in the novel object recognition test 24h post-administration in WT, but not in KO2A mice. The comparable effect of fluoxetine on extracellular 5-HT in the mPFC of both genotypes suggests that presynaptic differences are not accountable. In contrast, single unit recordings of mPFC putative pyramidal neurons showed that fluoxetine (1.8-7.2mg/kg i.v.) significantly increased neuronal discharge in KO2A but not in WT mice. This effect is possibly mediated by an altered excitatory/inhibitory balance in the PFC in KO2A mice. Overall, the present results suggest that 5-HT2A receptors play a detrimental role in long-term memory deficits mediated by an excess 5-HT in PFC.

  6. Multiple sclerosis lymphocytes upregulate A2A adenosine receptors that are antiinflammatory when stimulated.

    PubMed

    Vincenzi, Fabrizio; Corciulo, Carmen; Targa, Martina; Merighi, Stefania; Gessi, Stefania; Casetta, Ilaria; Gentile, Mauro; Granieri, Enrico; Borea, Pier Andrea; Varani, Katia

    2013-08-01

    Multiple sclerosis (MS) is an autoimmune-mediated inflammatory disease characterized by multifocal areas of demyelination. Experimental evidence indicates that A2A adenosine receptors (ARs) play a pivotal role in the inhibition of inflammatory processes. The aim of this study was to investigate the contribution of A2A ARs in the inhibition of key pro-inflammatory mediators for the pathogenesis of MS. In lymphocytes from MS patients, A1, A2A, A2B, and A3 ARs were analyzed by using RT-PCR, Western blotting, immunofluorescence, and binding assays. Moreover the effect of A2A AR stimulation on proinflammatory cytokine release such as TNF-α, IFN-γ, IL-6, IL-1β, IL-17, and on lymphocyte proliferation was evaluated. The capability of an A2A AR agonist on the modulation of very late antigen (VLA)-4 expression and NF-κB was also explored. A2A AR upregulation was observed in lymphocytes from MS patients in comparison with healthy subjects. The stimulation of these receptors mediated a significant inhibition of TNF-α, IFN-γ, IL-6, IL-1β, IL-17, and cell proliferation as well as VLA-4 expression and NF-κB activation. This new evidence highlights that A2A AR agonists could represent a novel therapeutic tool for MS treatment as suggested by the antiinflammatory role of A2A ARs in lymphocytes from MS patients.

  7. A covalent antagonist for the human adenosine A2A receptor.

    PubMed

    Yang, Xue; Dong, Guo; Michiels, Thomas J M; Lenselink, Eelke B; Heitman, Laura; Louvel, Julien; IJzerman, Ad P

    2016-12-03

    The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine.

  8. Functions, dysfunctions and possible therapeutic relevance of adenosine A2A receptors in Huntington's disease.

    PubMed

    Popoli, Patrizia; Blum, David; Martire, Alberto; Ledent, Catherine; Ceruti, Stefania; Abbracchio, Maria P

    2007-04-01

    The aim of this review is to summarize and critically discuss the complex role played by adenosine A(2A) receptors (A(2A)Rs) in Huntington's disease (HD). Since A(2A)Rs are mainly localized on the neurons, which degenerate early in HD, and given their ability to stimulate glutamate outflow and inflammatory gliosis, it was hypothesized that they could be involved in the pathogenesis of HD, and that A(2A)R antagonists could be neuroprotective. This was further sustained by the demonstration that A(2A)Rs and underlying signaling systems undergo profound changes in cellular and animal models of HD. More recently, however, the equation A(2A) receptor blockade=neuroprotection has appeared too simplistic. First, it is now definitely clear that, besides mediating 'bad' responses (for example, stimulation of glutamate outflow and excessive glial activation), A(2A)Rs also promote 'good' responses (such as trophic and antinflammatory effects). This implies that A(2A)R blockade results either in pro-toxic or neuroprotective effects according to the mechanisms involved in a given experimental model. Second, since HD is a chronically progressive disease, the multiple mechanisms involving A(2A)Rs may play different relative roles along the degenerative process. Such different mechanisms can be influenced by A(2A)R activation or blockade in different ways, even leading to opposite outcomes depending on the time of agonist/antagonist administration. The number, and the complexity, of the possible scenarios is further increased by the influence of mutant Huntingtin on both the expression and functions of A(2A)Rs, and by the strikingly different effects mediated by A(2A)Rs expressed by different cell populations within the brain.

  9. Activation of NR2A receptors induces ischemic tolerance through CREB signaling.

    PubMed

    Terasaki, Yasukazu; Sasaki, Tsutomu; Yagita, Yoshiki; Okazaki, Shuhei; Sugiyama, Yukio; Oyama, Naoki; Omura-Matsuoka, Emi; Sakoda, Saburo; Kitagawa, Kazuo

    2010-08-01

    Previous exposure to a nonlethal ischemic insult protects the brain against subsequent harmful ischemia. N-methyl-D-aspartate (NMDA) receptors are a highly studied target of neuroprotection after ischemia. Recently, NMDA receptor subtypes were implicated in neuronal survival and death. We focused on the contribution of NR2A and cyclic-AMP response element (CRE)-binding protein (CREB) signaling to ischemic tolerance using primary cortical neurons. Ischemia in vitro was modeled by oxygen-glucose deprivation (OGD). Ischemic tolerance was induced by applying 45-mins OGD 24 h before 180-mins OGD. Sublethal OGD also induced cross-tolerance against lethal glutamate and hydrogen peroxide. After sublethal OGD, expression of phosphorylated CREB and CRE transcriptional activity were significantly increased. When CRE activity was inhibited by CREB-S133A, a mutant CREB, ischemic tolerance was abolished. Inhibiting NR2A using NVP-AAM077 attenuated preconditioning-induced neuroprotection and correlated with decreased CRE activity levels. Activating NR2A using bicuculline and 4-aminopiridine induced resistance to lethal ischemia accompanied by elevated CRE activity levels, and this effect was abolished by NVP-AAM077. Elevated brain-derived neurotrophic factor (BDNF) transcriptional activities were observed after sublethal OGD and administration of bicuculline and 4-aminopiridine. NR2A-containing NMDA receptors and CREB signaling have important functions in the induction of ischemic tolerance. This may provide potential novel therapeutic strategies to treat ischemic stroke.

  10. Expression and Function of Serotonin 2A and 2B Receptors in the Mammalian Respiratory Network

    PubMed Central

    Koch, Uwe R.; Bischoff, Anna-Maria; Kron, Miriam; Bock, Nathalie; Manzke, Till

    2011-01-01

    Neurons of the respiratory network in the lower brainstem express a variety of serotonin receptors (5-HTRs) that act primarily through adenylyl cyclase. However, there is one receptor family including 5-HT2A, 5-HT2B, and 5-HT2C receptors that are directed towards protein kinase C (PKC). In contrast to 5-HT2ARs, expression and function of 5-HT2BRs within the respiratory network are still unclear. 5-HT2BR utilizes a Gq-mediated signaling cascade involving calcium and leading to activation of phospholipase C and IP3/DAG pathways. Based on previous studies, this signal pathway appears to mediate excitatory actions on respiration. In the present study, we analyzed receptor expression in pontine and medullary regions of the respiratory network both at the transcriptional and translational level using quantitative RT-PCR and self-made as well as commercially available antibodies, respectively. In addition we measured effects of selective agonists and antagonists for 5-HT2ARs and 5-HT2BRs given intra-arterially on phrenic nerve discharges in juvenile rats using the perfused brainstem preparation. The drugs caused significant changes in discharge activity. Co-administration of both agonists revealed a dominance of the 5-HT2BR. Given the nature of the signaling pathways, we investigated whether intracellular calcium may explain effects observed in the respiratory network. Taken together, the results of this study suggest a significant role of both receptors in respiratory network modulation. PMID:21789169

  11. Sleep Deprivation Increases Cerebral Serotonin 2A Receptor Binding in Humans

    PubMed Central

    Elmenhorst, David; Kroll, Tina; Matusch, Andreas; Bauer, Andreas

    2012-01-01

    Study Objectives: Serotonin and its cerebral receptors play an important role in sleep-wake regulation. The aim of the current study is to investigate the effect of 24-h total sleep deprivation on the apparent serotonin 2A receptor (5-HT2AR) binding capacity in the human brain to test the hypothesis that sleep deprivation induces global molecular alterations in the cortical serotonergic receptor system. Design: Volunteers were tested twice with the subtype-selective radiotracer [18F]altanserin and positron emission tomography (PET) for imaging of 5-HT2ARs at baseline and after 24 h of sleep deprivation. [18F]Altanserin binding potentials were analyzed in 13 neocortical regions of interest. The efficacy of sleep deprivation was assessed by questionnaires, waking electroencephalography, and cognitive performance measurements. Setting: Sleep laboratory and neuroimaging center. Patients or Participants: Eighteen healthy volunteers. Interventions: Sleep deprivation. Measurements and Results: A total of 24 hours of sleep deprivation led to a 9.6% increase of [18F]altanserin binding on neocortical 5-HT2A receptors. Significant region-specific increases were found in the medial inferior frontal gyrus, insula, and anterior cingulate, parietal, sensomotoric, and ventrolateral prefrontal cortices. Conclusions: This study demonstrates that a single night of total sleep deprivation causes significant increases of 5-HT2AR binding potentials in a variety of cortical regions although the increase declines as sleep deprivation continued. It provides in vivo evidence that total sleep deprivation induces adaptive processes in the serotonergic system of the human brain. Citation: Elmenhorst D; Kroll T; Matusch A; Bauer A. Sleep Deprivation Increases Cerebral Serotonin 2A Receptor Binding in Humans. SLEEP 2012;35(12):1615-1623. PMID:23204604

  12. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

    SciTech Connect

    Watanabe, Kanako; Kanno, Takeshi; Oshima, Tadayuki; Miwa, Hiroto; Tashiro, Chikara; Nishizaki, Tomoyuki

    2008-03-07

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-D-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G{sub 1} phase of cell cycling and decreased the proportion in the S/G{sub 2} phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G{sub 1} phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling.

  13. Prosurvival NMDA 2A receptor signaling mediates postconditioning neuroprotection in the hippocampus.

    PubMed

    Zhang, Xi; Zhang, Quanguang; Tu, Jingyi; Zhu, Ying; Yang, Fang; Liu, Bin; Brann, Darrell; Wang, Ruimin

    2015-03-01

    Ischemic postconditioning (Post C), which involves administration of a brief ischemia after the initial ischemic event, has been demonstrated to be strongly neuroprotective against global cerebral ischemia (GCI) and to improve cognitive outcome. To enhance understanding of the underlying mechanisms, the current study examined the role of NMDA receptors in mediating the beneficial effects of Post C (3 min ischemia) administered 2 days after GCI in adult male rats. The results revealed that Post C was strongly neuroprotective against GCI, and that this effect was blocked by administration of the NMDA receptor antagonist MK-801. Further work revealed that the NR2A-type NMDA receptors mediate the Post C beneficial effects as administration of a NR2A-preferring antagonist (NVP-A) blocked Post C neuroprotection and cognitive enhancement, while administration of a NR2B-preferring antagonist (Ro25) was without effect. Post C significantly up-regulated NR2A levels and phosphorylation of NR2A in the hippocampal CA1 region after Post C. Post C also increased Ca(2+) influx and activation/phosphorylation of CamKIIα at Thr(286), effects that were NR2A mediated as they were blocked by NVP-A. Phosphorylation of ERK and CREB was also increased by Post C, as were two downstream CREB-dependent prosurvival factors, brain derived neurotropic factor (BDNF) and Bcl2, effects that were blocked by the NR2A antagonist, NVP-A. Taken as a whole, the current study provides evidence that NR2A-activation and downstream prosurvival signaling is a critical mediator of Post C-induced neuroprotection and cognitive enhancement following GCI.

  14. Effects of serotonin 2A/1A receptor stimulation on social exclusion processing.

    PubMed

    Preller, Katrin H; Pokorny, Thomas; Hock, Andreas; Kraehenmann, Rainer; Stämpfli, Philipp; Seifritz, Erich; Scheidegger, Milan; Vollenweider, Franz X

    2016-05-03

    Social ties are crucial for physical and mental health. However, psychiatric patients frequently encounter social rejection. Moreover, an increased reactivity to social exclusion influences the development, progression, and treatment of various psychiatric disorders. Nevertheless, the neuromodulatory substrates of rejection experiences are largely unknown. The preferential serotonin (5-HT) 2A/1A receptor agonist, psilocybin (Psi), reduces the processing of negative stimuli, but whether 5-HT2A/1A receptor stimulation modulates the processing of negative social interactions remains unclear. Therefore, this double-blind, randomized, counterbalanced, cross-over study assessed the neural response to social exclusion after the acute administration of Psi (0.215 mg/kg) or placebo (Pla) in 21 healthy volunteers by using functional magnetic resonance imaging (fMRI) and resting-state magnetic resonance spectroscopy (MRS). Participants reported a reduced feeling of social exclusion after Psi vs. Pla administration, and the neural response to social exclusion was decreased in the dorsal anterior cingulate cortex (dACC) and the middle frontal gyrus, key regions for social pain processing. The reduced neural response in the dACC was significantly correlated with Psi-induced changes in self-processing and decreased aspartate (Asp) content. In conclusion, 5-HT2A/1A receptor stimulation with psilocybin seems to reduce social pain processing in association with changes in self-experience. These findings may be relevant to the normalization of negative social interaction processing in psychiatric disorders characterized by increased rejection sensitivity. The current results also emphasize the importance of 5-HT2A/1A receptor subtypes and the Asp system in the control of social functioning, and as prospective targets in the treatment of sociocognitive impairments in psychiatric illnesses.

  15. Effects of serotonin 2A/1A receptor stimulation on social exclusion processing

    PubMed Central

    Preller, Katrin H.; Pokorny, Thomas; Hock, Andreas; Kraehenmann, Rainer; Stämpfli, Philipp; Seifritz, Erich; Scheidegger, Milan; Vollenweider, Franz X.

    2016-01-01

    Social ties are crucial for physical and mental health. However, psychiatric patients frequently encounter social rejection. Moreover, an increased reactivity to social exclusion influences the development, progression, and treatment of various psychiatric disorders. Nevertheless, the neuromodulatory substrates of rejection experiences are largely unknown. The preferential serotonin (5-HT) 2A/1A receptor agonist, psilocybin (Psi), reduces the processing of negative stimuli, but whether 5-HT2A/1A receptor stimulation modulates the processing of negative social interactions remains unclear. Therefore, this double-blind, randomized, counterbalanced, cross-over study assessed the neural response to social exclusion after the acute administration of Psi (0.215 mg/kg) or placebo (Pla) in 21 healthy volunteers by using functional magnetic resonance imaging (fMRI) and resting-state magnetic resonance spectroscopy (MRS). Participants reported a reduced feeling of social exclusion after Psi vs. Pla administration, and the neural response to social exclusion was decreased in the dorsal anterior cingulate cortex (dACC) and the middle frontal gyrus, key regions for social pain processing. The reduced neural response in the dACC was significantly correlated with Psi-induced changes in self-processing and decreased aspartate (Asp) content. In conclusion, 5-HT2A/1A receptor stimulation with psilocybin seems to reduce social pain processing in association with changes in self-experience. These findings may be relevant to the normalization of negative social interaction processing in psychiatric disorders characterized by increased rejection sensitivity. The current results also emphasize the importance of 5-HT2A/1A receptor subtypes and the Asp system in the control of social functioning, and as prospective targets in the treatment of sociocognitive impairments in psychiatric illnesses. PMID:27091970

  16. Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies

    DTIC Science & Technology

    2012-12-01

    in Traumatic Optic Neuropathies ” PRINCIPAL INVESTIGATOR: Gregory I. Liou, PhD CONTRACTING ORGANIZATION: Georgia Health Sciences...Adenosine Receptor A2A in Traumatic Optic Neuropathies 5b. GRANT NUMBER W81XWH-11-2-0046 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT...ABSTRACT Our goal is to develop an early therapeutic intervention before the progression of traumatic optic neuropathy (TON), a vision-threatening

  17. Key modulatory role of presynaptic adenosine A2A receptors in cortical neurotransmission to the striatal direct pathway.

    PubMed

    Quiroz, César; Luján, Rafael; Uchigashima, Motokazu; Simoes, Ana Patrícia; Lerner, Talia N; Borycz, Janusz; Kachroo, Anil; Canas, Paula M; Orru, Marco; Schwarzschild, Michael A; Rosin, Diane L; Kreitzer, Anatol C; Cunha, Rodrigo A; Watanabe, Masahiko; Ferré, Sergi

    2009-11-18

    Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D1 and D2 receptors, respectively. Adenosine A2A receptors are considered novel antiparkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D2 receptor function. The present study provides evidence for the existence of an additional, functionally significant, segregation of A2A receptors at the presynaptic level. Using integrated anatomical, electrophysiological, and biochemical approaches, we demonstrate that presynaptic A2A receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of corticostriatal neurotransmission. Presynaptic striatal A2A receptors could provide a new target for the treatment of neuropsychiatric disorders.

  18. 5-HT2A/C receptors mediate the antipsychotic-like effects of alstonine.

    PubMed

    Linck, V M; Bessa, M M; Herrmann, A P; Iwu, M M; Okunji, C O; Elisabetsky, E

    2012-01-10

    The purpose of this study was to determine the effects of alstonine, an indole alkaloid with putative antipsychotic effects, on working memory by using the step-down inhibitory avoidance paradigm and MK801-induced working memory deficits in mice. Additionally, the role of serotonin 5-HT2A/C receptors in the effects of alstonine on mouse models associated with positive (MK801-induced hyperlocomotion), negative (MK801-induced social interaction deficit), and cognitive (MK801-induced working memory deficit) schizophrenia symptoms was examined. Treatment with alstonine was able to prevent MK801-induced working memory deficit, indicating its potential benefit for cognitive deficits now seen as a core symptom in the disease. Corroborating previously reported data, alstonine was also effective in counteracting MK801-induced hyperlocomotion and social interaction deficit. Ritanserin, a 5-HT2A/C receptor antagonist, prevented alstonine's effects on these three behavioral parameters. This study presents additional evidence that 5-HT2A/C receptors are central to the antipsychotic-like effects of alstonine, consistently seen in mouse models relevant to the three dimensions of schizophrenia symptoms.

  19. Polymorphism in the Serotonin Receptor 2a (HTR2A) Gene as Possible Predisposal Factor for Aggressive Traits

    PubMed Central

    Banlaki, Zsofia; Elek, Zsuzsanna; Nanasi, Tibor; Szekely, Anna; Nemoda, Zsofia; Sasvari-Szekely, Maria; Ronai, Zsolt

    2015-01-01

    Aggressive manifestations and their consequences are a major issue of mankind, highlighting the need for understanding the contributory factors. Still, aggression-related genetic analyses have so far mainly been conducted on small population subsets such as individuals suffering from a certain psychiatric disorder or a narrow-range age cohort, but no data on the general population is yet available. In the present study, our aim was to identify polymorphisms in genes affecting neurobiological processes that might explain some of the inter-individual variation between aggression levels in the non-clinical Caucasian adult population. 55 single nucleotide polymorphisms (SNP) were simultaneously determined in 887 subjects who also filled out the self-report Buss-Perry Aggression Questionnaire (BPAQ). Single marker association analyses between genotypes and aggression scores indicated a significant role of rs7322347 located in the HTR2A gene encoding serotonin receptor 2a following Bonferroni correction for multiple testing (p = 0.0007) both for males and females. Taking the four BPAQ subscales individually, scores for Hostility, Anger and Physical Aggression showed significant association with rs7322347 T allele in themselves, while no association was found with Verbal Aggression. Of the subscales, relationship with rs7322347 was strongest in the case of Hostility, where statistical significance virtually equaled that observed with the whole BPAQ. In conclusion, this is the first study to our knowledge analyzing SNPs in a wide variety of genes in terms of aggression in a large sample-size non-clinical adult population, also describing a novel candidate polymorphism as predisposal to aggressive traits. PMID:25658328

  20. The G2A receptor (GPR132) contributes to oxaliplatin-induced mechanical pain hypersensitivity.

    PubMed

    Hohmann, Stephan W; Angioni, Carlo; Tunaru, Sorin; Lee, Seungkyu; Woolf, Clifford J; Offermanns, Stefan; Geisslinger, Gerd; Scholich, Klaus; Sisignano, Marco

    2017-03-27

    Chemotherapy-induced peripheral neuropathic pain (CIPN) is a common and severe debilitating side effect of many widely used cytostatics. However, there is no approved pharmacological treatment for CIPN available. Among other substances, oxaliplatin causes CIPN in up to 80% of treated patients. Here, we report the involvement of the G-protein coupled receptor G2A (GPR132) in oxaliplatin-induced neuropathic pain in mice. We found that mice deficient in the G2A-receptor show decreased mechanical hypersensitivity after oxaliplatin treatment. Lipid ligands of G2A were found in increased concentrations in the sciatic nerve and dorsal root ganglia of oxaliplatin treated mice. Calcium imaging and patch-clamp experiments show that G2A activation sensitizes the ligand-gated ion channel TRPV1 in sensory neurons via activation of PKC. Based on these findings, we conclude that targeting G2A may be a promising approach to reduce oxaliplatin-induced TRPV1-sensitization and the hyperexcitability of sensory neurons and thereby to reduce pain in patients treated with this chemotherapeutic agent.

  1. Clinical/pharmacological aspect of adenosine A2A receptor antagonist for dyskinesia.

    PubMed

    Kanda, Tomoyuki; Uchida, Shin-ichi

    2014-01-01

    Dopamine replacement therapy using the dopamine precursor, l-3,4-dihydroxyphenylalanine (l-DOPA), with a peripheral dopa decarboxylase inhibitor is the most effective treatment currently available for the symptoms of Parkinson's disease (PD). However, the long-term use of dopaminergic therapies for PD is often limited by the development of motor response complications, such as dyskinesia. Adenosine A2A receptors are a promising nondopaminergic target for the treatment of PD. The treatment of motor response complications involves combinations of regular and controlled release L-DOPA, perhaps with the addition of a COMT inhibitor or the use of a longer-acting dopamine agonist. However, when dyskinesia is already established, the increase in dopaminergic load produced by the addition of a dopamine agonist can result in an increase in the severity and duration of dyskinesia. Currently, there are no well-tolerated antidyskinesia agents available. Amantadine, which may exert its effects through the inhibition of N-methyl-D-aspartate (NMDA) receptors, shows some effects on established dyskinesia. Dyskinesia has a negative impact on the quality of life of patients, sometimes being more disabling than PD itself. Although some patients prefer experiencing dyskinesia than being in the OFF state and unable to move, alternative, more effective therapies are still required for severe disabling dyskinesia to afford patients an improved quality of life while in the ON state. The mechanisms causing and maintaining the dyskinesia have not been clarified. The application of a nondopaminergic approach to modify the basal ganglial activity would be helpful to better understand and treat dyskinesia. The use of an adenosine A2A receptor may provide one such approach. In this literature review, we will summarize the current knowledge from both clinical and nonclinical studies on the effects of adenosine A2A receptor blockade on dyskinesia.

  2. Selective 5HT2A and 5HT6 Receptor Antagonists Promote Sleep in Rats

    PubMed Central

    Morairty, Stephen R.; Hedley, Linda; Flores, Judith; Martin, Renee; Kilduff, Thomas S.

    2008-01-01

    Study Objectives: Serotonin (5-HT) has long been implicated in the control of sleep and wakefulness. This study evaluated the hypnotic efficacy of the 5-HT6 antagonist RO4368554 (RO) and the 5-HT2A receptor antagonist MDL100907 (MDL) relative to zolpidem. Design: A randomized, repeated-measures design was utilized in which Wistar rats received intraperitoneal injections of RO (1.0, 3.0, and 10 mg/kg), MDL (0.1, 1.0 and 3.0 mg/kg), zolpidem (10 mg/kg), or vehicle in the middle of the dark (active) period. Electroencephalogram, electromyogram, body temperature (Tb) and locomotor activity were analyzed for 6 hours after injection. Measurements and Results: RO, MDL, and zolpidem all produced significant increases in sleep and decreases in waking, compared with vehicle control. All 3 doses of MDL produced more consolidated sleep, increased non-rapid eye movement sleep (NREM) sleep, and increased electroencephalographic delta power during NREM sleep. The highest dose of RO (10.0 mg/kg) produced significant increases in sleep and decreases in waking during hour 2 following dosing. These increases in sleep duration were associated with greater delta power during NREM sleep. ZO Zolpidem induced sleep with the shortest latency and significantly increased NREM sleep and delta power but also suppressed rapid eye movement sleep sleep; in contrast, neither RO nor MDL affected rapid eye movement sleep. Whereas RO did not affect Tb, both zolpidem and MDL reduced Tb relative to vehicle-injected controls. Conclusions: These results support a role for 5-HT2A receptor modulation in NREM sleep and suggest a previously unrecognized role for 5-HT6 receptors in sleep-wake regulation. Citation: Morairty SR; Hedley L; Flores J; Martin R; Kilduff TS. Selective 5HT2A and 5HT6 receptor antagonists promote sleep in rats. SLEEP 2008;31(1):34-44. PMID:18220076

  3. The role of serotonin 5-HT2A receptors in memory and cognition

    PubMed Central

    Zhang, Gongliang; Stackman, Robert W.

    2015-01-01

    Serotonin 5-HT2A receptors (5-HT2ARs) are widely distributed in the central nervous system, especially in brain region essential for learning and cognition. In addition to endogenous 5-HT, several hallucinogens, antipsychotics, and antidepressants function by targeting 5-HT2ARs. Preclinical studies show that 5-HT2AR antagonists have antipsychotic and antidepressant properties, whereas agonist ligands possess cognition-enhancing and hallucinogenic properties. Abnormal 5-HT2AR activity is associated with a number of psychiatric disorders and conditions, including depression, schizophrenia, and drug addiction. In addition to its traditional activity as a G protein-coupled receptor (GPCR), recent studies have defined novel operations of 5-HT2ARs. Here we review progress in the (1) receptor anatomy and biology: distribution, signaling, polymerization and allosteric modulation; and (2) receptor functions: learning and memory, hallucination and spatial cognition, and mental disorders. Based on the recent progress in basic research on the 5-HT2AR, it appears that post-training 5-HT2AR activation enhances non-spatial memory consolidation, while pre-training 5-HT2AR activation facilitates fear extinction. Further, the potential influence that 5-HT2AR-elicited visual hallucinations may have on visual cue (i.e., landmark) guided spatial cognition is discussed. We conclude that the development of selective 5-HT2AR modulators to target distinct signaling pathways and neural circuits represents a new possibility for treating emotional, neuropsychiatric, and neurodegenerative disorders. PMID:26500553

  4. Ultraslow Water-Mediated Transmembrane Interactions Regulate the Activation of A2A Adenosine Receptor

    NASA Astrophysics Data System (ADS)

    Lee, Yoonji; Kim, Songmi; Choi, Sun; Hyeon, Changbong

    2016-09-01

    Water molecules inside G-protein coupled receptor have recently been spotlighted in a series of crystal structures. To decipher the dynamics and functional roles of internal waters in GPCR activity, we studied A$_{\\text{2A}}$ adenosine receptor using $\\mu$sec-molecular dynamics simulations. Our study finds that the amount of water flux across the transmembrane (TM) domain varies depending on the receptor state, and that the water molecules of the TM channel in the active state flow three times slower than those in the inactive state. Depending on the location in solvent-protein interface as well as the receptor state, the average residence time of water in each residue varies from $\\sim\\mathcal{O}(10^2)$ psec to $\\sim\\mathcal{O}(10^2)$ nsec. Especially, water molecules, exhibiting ultraslow relaxation ($\\sim\\mathcal{O}(10^2)$ nsec) in the active state, are found around the microswitch residues that are considered activity hotspots for GPCR function. A continuous allosteric network spanning the TM domain, arising from water-mediated contacts, is unique in the active state, underscoring the importance of slow waters in the GPCR activation.

  5. Disrupting 5-HT2A Receptor/PDZ Protein Interactions Reduces Hyperalgesia and Enhances SSRI Efficacy in Neuropathic Pain

    PubMed Central

    Pichon, Xavier; Wattiez, Anne S; Becamel, Carine; Ehrlich, Ingrid; Bockaert, Joel; Eschalier, Alain; Marin, Philippe; Courteix, Christine

    2010-01-01

    Antidepressants are one of the first-line treatments for neuropathic pain. Despite the influence of serotonin (5-hydroxytryptamine, 5-HT) in pain modulation, selective serotonin reuptake inhibitors (SSRIs) are less effective than tricyclic antidepressants. Here, we show, in diabetic neuropathic rats, an alteration of the antihyperalgesic effect induced by stimulation of 5-HT2A receptors, which are known to mediate SSRI-induced analgesia. 5-HT2A receptor density was not changed in the spinal cord of diabetic rats, whereas postsynaptic density protein-95 (PSD-95), one of the PSD-95/disc large suppressor/zonula occludens-1 (PDZ) domain containing proteins interacting with these receptors, was upregulated. Intrathecal injection of a cell-penetrating peptidyl mimetic of the 5-HT2A receptor C-terminus, which disrupts 5-HT2A receptor–PDZ protein interactions, induced an antihyperalgesic effect in diabetic rats, which results from activation of 5-HT2A receptors by endogenous 5-HT. The peptide also enhanced antihyperalgesia induced by the SSRI fluoxetine. Its effects likely resulted from an increase in receptor responsiveness, because it revealed functional 5-HT2A receptor-operated Ca2+ responses in neurons, an effect mimicked by knockdown of PSD-95. Hence, 5-HT2A receptor/PDZ protein interactions might contribute to the resistance to SSRI-induced analgesia in painful diabetic neuropathy. Disruption of these interactions might be a valuable strategy to design novel treatments for neuropathic pain and to increase the effectiveness of SSRIs. PMID:20531396

  6. Integrating Pharmacophore into Membrane Molecular Dynamics Simulations to Improve Homology Modeling of G Protein-coupled Receptors with Ligand Selectivity: A2A Adenosine Receptor as an Example.

    PubMed

    Zeng, Lingxiao; Guan, Mengxin; Jin, Hongwei; Liu, Zhenming; Zhang, Liangren

    2015-12-01

    Homology modeling has been applied to fill in the gap in experimental G protein-coupled receptors structure determination. However, achievement of G protein-coupled receptors homology models with ligand selectivity remains challenging due to structural diversity of G protein-coupled receptors. In this work, we propose a novel strategy by integrating pharmacophore and membrane molecular dynamics (MD) simulations to improve homology modeling of G protein-coupled receptors with ligand selectivity. To validate this integrated strategy, the A2A adenosine receptor (A2A AR), whose structures in both active and inactive states have been established, has been chosen as an example. We performed blind predictions of the active-state A2A AR structure based on the inactive-state structure and compared the performance of different refinement strategies. The blind prediction model combined with the integrated strategy identified ligand-receptor interactions and conformational changes of key structural elements related to the activation of A2 A AR, including (i) the movements of intracellular ends of TM3 and TM5/TM6; (ii) the opening of ionic lock; (iii) the movements of binding site residues. The integrated strategy of pharmacophore with molecular dynamics simulations can aid in the optimization in the identification of side chain conformations in receptor models. This strategy can be further investigated in homology modeling and expand its applicability to other G protein-coupled receptor modeling, which should aid in the discovery of more effective and selective G protein-coupled receptor ligands.

  7. Effect of low frequency electromagnetic fields on A2A adenosine receptors in human neutrophils

    PubMed Central

    Varani, Katia; Gessi, Stefania; Merighi, Stefania; Iannotta, Valeria; Cattabriga, Elena; Spisani, Susanna; Cadossi, Ruggero; Borea, Pier Andrea

    2002-01-01

    The present study describes the effect of low frequency, low energy, pulsing electromagnetic fields (PEMFs) on A2A adenosine receptors in human neutrophils.Saturation experiments performed using a high affinity adenosine antagonist [3H]-ZM 241385 revealed a single class of binding sites in control and in PEMF-treated human neutrophils with similar affinity (KD=1.05±0.10 and 1.08±0.12 nM, respectively). Furthermore, after 1 h of exposure to PEMFs the receptor density was statistically increased (P<0.01) (Bmax =126±10 and 215±15 fmol mg−1 protein, respectively).The effect of PEMFs was specific to the A2A adenosine receptors. This effect was also intensity, time and temperature dependent.In the adenylyl cyclase assays the A2A receptor agonists, HE-NECA and NECA, increased cyclic AMP accumulation in untreated human neutrophils with an EC50 value of 43 (40 – 47) and 255 (228 – 284) nM, respectively. The capability of HE-NECA and NECA to stimulate cyclic AMP levels in human neutrophils was increased (P<0.01) after exposure to PEMFs with an EC50 value of 10(8 – 13) and 61(52 – 71) nM, respectively.In the superoxide anion (O2−) production assays HE-NECA and NECA inhibited the generation of O2− in untreated human neutrophils, with an EC50 value of 3.6(3.1 – 4.2) and of 23(20 – 27) nM, respectively. Moreover, in PEMF-treated human neutrophils, the same compounds show an EC50 value of 1.6(1.2 – 2.1) and of 6.0(4.7 – 7.5) nM respectively.These results indicate the presence of significant alterations in the expression and in the functionality of adenosine A2A receptors in human neutrophils treated with PEMFs. PMID:11976268

  8. Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms

    PubMed Central

    Bhattacharyya, Samarjit; Puri, Sapna; Miledi, Ricardo; Panicker, Mitradas M.

    2002-01-01

    Serotonin (5-HT), a major neurotransmitter, has a large number of G protein-coupled receptors in mammals. On activation by exposure to their ligand, 5-HT2 receptor subtypes increase IP3 levels and undergo desensitization and internalization. To visualize the receptor in cells during these processes, we have constructed a 5-HT2A-enhanced GFP (SR2-GFP) fusion receptor. We show that this fusion receptor undergoes internalization on exposure to its natural ligand, 5-HT. Because 5-HT2A receptors activate the phospholipase C pathway, we studied the effect of protein kinase C (PKC) on the internalization process and found that activation of PKC by its specific activator phorbol 12-myristate 13-acetate, in the absence of 5-HT, leads to internalization of the receptor. Moreover, inhibition of PKC by its inhibitor sphingosine in the presence of 5-HT prevents the internalization process, suggesting that activation of PKC is sufficient and necessary for the internalization of 5-HT2A receptors. We also show that SR2-GFP recycles back to the plasma membrane after 5-HT-dependent internalization, suggesting a mechanism for resensitization. In addition, receptors that have been internalized on addition of phorbol 12-myristate 13-acetate in the absence of 5-HT also recycle to the surface, with a time course similar to that seen after activation of the receptors by 5-HT. Our study suggests that 5-HT2A receptors internalize and return to the surface after both serotonin- and PKC-mediated processes. This study reveals a role for PKC in receptor internalization and also shows that 5-HT2A receptors are recycled. PMID:12388782

  9. Potential therapeutic relevance of adenosine A2B and A2A receptors in the central nervous system.

    PubMed

    Popoli, Patrizia; Pepponi, Rita

    2012-09-01

    Adenosine A2B and, much more importantly, adenosine A2A receptors modulate many physiological and pathological processes in the brain. In this review, the most recent evidence concerning the role of such receptors and their potential therapeutic relevance is discussed. The low affinity of A2B receptors for adenosine implies that they might represent a good therapeutic target, since they are activated only under pathological conditions (when adenosine levels raise up to micromolar concentrations). The availability of selective ligands for A2B receptors would allow exploration of such an hypothesis. Since adenosine A2A receptors mediate both potentially neuroprotective and potentially neurotoxic effects, their role in neurodegenerative diseases is highly controversial. Nevertheless, A2A receptor antagonists have shown clear antiparkinsonian effects, and a great interest exists on the role of A2A receptors in Alzheimer's disease, brain ischaemia, spinal cord injury, drug addiction and other conditions. In order to establish whether such receptors represent a target for CNS diseases, at least two conditions are needed: the full comprehension of A2A-dependent mechanisms and the availability of ligands capable of discriminating among the different receptor populations.

  10. Down-regulation of adenosine A1 and A2A receptors in peripheral cells from idiopathic normal-pressure hydrocephalus patients.

    PubMed

    Casati, Martina; Arosio, Beatrice; Gussago, Cristina; Ferri, Evelyn; Magni, Lorenzo; Assolari, Lara; Scortichini, Valeria; Nani, Carolina; Rossi, Paolo Dionigi; Mari, Daniela

    2016-02-15

    Idiopathic normal-pressure hydrocephalus (iNPH) is a neurological disease that usually develops in the elderly. Natural history of iNPH is still unknown. It has been hypothesized that cerebrovascular diseases could have a role in etiology of chronic hydrocephalus and studies show an increased prevalence of cardiovascular diseases in iNPH patients. Moreover, evidences show a possible alteration of immune system in iNPH patients. Adenosine (Ado) is a metabolite produced in response to metabolic stress and injury. Adenosine and its receptors play an important role in vascular protection and in the modulation of inflammatory reactions and neuroinflammation. Our aim is to evaluate gene and protein expression of A1R and A2AR in the peripheral blood mononuclear cells (PBMCs) from iNPH patients compared to control subjects. We investigate if Ado system, that plays an important role in central nervous system, in vascular system, and also in inflammation, is involved in pathophysiology of iNPH disease. Our analysis showed that A1R mRNA levels and A1R density in PBMCs from iNPH patients were significantly lower than CT subjects (0.84 ± 0.12 and 2.42 ± 0.42, p<0.001 and 0.31 ± 0.02 and 0.42 ± 0.04, p=0.043; respectively). About A2AR, the gene expression in PBMCs was significantly lower in iNPH than CT (0.65 ± 0.09 and 1.5 ± 0.14, p<0.001) as well as there was a trend in protein expression: iNPH and CT (0.51 ± 0.05 and 0.62 ± 0.03; p=0.172). This preliminary study underlines the involvement of Ado system in iNPH disease whose pathophysiology is still unclear.

  11. Gender, personality, and serotonin-2A receptor binding in healthy subjects

    PubMed Central

    Soloff, Paul H.; Price, Julie C.; Mason, Neale Scott; Becker, Carl; Meltzer, Carolyn C.

    2009-01-01

    The vulnerability to mood disorders, impulsive-aggression, eating disorders, and suicidal behavior varies greatly with gender, and may reflect gender differences in central serotonergic function. We investigated the relationships of gender, mood, impulsivity, aggression and temperament to 5HT2A receptor binding in 21 healthy subjects using [18F]altanserin and PET neuro-imaging. Binding potentials in pre-defined Regions of Interest (ROI) were calculated using the Logan graphical method, corrected for partial volume effects, and compared by gender with age co-varied. SPM analysis was used for voxel level comparisons. Altanserin binding (BPp) was greater in male than female subjects in 9 ROIs: hippocampus (HIP) and Lt. HIP, lateral orbital frontal cortex (LOF) and Lt.LOF, left medial frontal cortex (Lt.MFC), left medial temporal cortex (Lt. MTC), left occipital cortex (Lt. OCC), thalamus (THL) and Lt. THL. Differences in Lt. HIP and Lt. MTL remained significant after Bonferroni correction. Gender differences were noted in the co-variation of psychological traits with BPp values in specific ROIs. Among males alone, aggression was negatively correlated with BPp values in Lt. LOF and Lt. MFC, and Suspiciousness positively correlated in LOF, Lt. LOF and Lt. MFC. Among female subjects alone, Negativism was positively correlated with BPp values in HIP, and Verbal Hostility in Lt. HIP. Altanserin binding in Lt. MTC was positively correlated with Persistence, with no significant gender effect. Gender differences in 5HT2A receptor function in specific ROIs may mediate expression of psychological characteristics such as aggression, suspiciousness and negativism. Future studies of 5HT2A receptor function and its relationship to behavior should control for gender. PMID:19959344

  12. Physical origins of remarkable thermostabilization by an octuple mutation for the adenosine A2a receptor

    NASA Astrophysics Data System (ADS)

    Kajiwara, Yuta; Ogino, Takahiro; Yasuda, Satoshi; Takamuku, Yuuki; Murata, Takeshi; Kinoshita, Masahiro

    2016-07-01

    It was experimentally showed that the thermal stability of a membrane protein, the adenosine A2a receptor, was remarkably enhanced by an octuple mutation. Here we theoretically prove that the energy decrease arising from the formation of protein intramolecular hydrogen bonds and the solvent-entropy gain upon protein folding are made substantially larger by the mutation, leading to the remarkable enhancement. The solvent is formed by hydrocarbon groups constituting nonpolar chains of the lipid bilayer within a membrane. The mutation modifies geometric characteristics of the structure so that the solvent crowding can be reduced to a larger extent when the protein folds.

  13. Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies (Addendum)

    DTIC Science & Technology

    2016-03-01

    diabetic retinopathy. Life Sci. 2013 Jul 30;93(2-3):78-88. doi: 10.1016/j.lfs.2013.05.024. Epub 2013 Jun 12.PMID:23770229 7 AIMS: This study was...undertaken to determine the effect of an adenosine kinase inhibitor (AKI) in diabetic retinopathy (DR). We have shown previously that adenosine signaling...via A2A receptors (A2AAR) is involved in retinal protection from diabetes -induced inflammation. Here we demonstrate that AKI-enhanced adenosine

  14. Protein phosphatase 2A regulates interleukin-2 receptor complex formation and JAK3/STAT5 activation.

    PubMed

    Ross, Jeremy A; Cheng, Hanyin; Nagy, Zsuzsanna S; Frost, Jeffrey A; Kirken, Robert A

    2010-02-05

    Reversible protein phosphorylation plays a key role in interleukin-2 (IL-2) receptor-mediated activation of Janus tyrosine kinase 3 (JAK3) and signal transducer and activator of transcription 5 (STAT5) in lymphocytes. Although the mechanisms governing IL-2-induced tyrosine phosphorylation and activation of JAK3/STAT5 have been extensively studied, the role of serine/threonine phosphorylation in controlling these effectors remains to be elucidated. Using phosphoamino acid analysis, JAK3 and STAT5 were determined to be serine and tyrosine-phosphorylated in response to IL-2 stimulation of the human natural killer-like cell line, YT. IL-2 stimulation also induced serine/threonine phosphorylation of IL-2Rbeta, but not IL-2Rgamma. To investigate the regulation of serine/threonine phosphorylation in IL-2 signaling, the roles of protein phosphatase 1 (PP1) and 2A (PP2A) were examined. Inhibition of phosphatase activity by calyculin A treatment of YT cells resulted in a significant induction of serine phosphorylation of JAK3 and STAT5, and serine/threonine phosphorylation of IL-2Rbeta. Moreover, inhibition of PP2A, but not PP1, diminished IL-2-induced tyrosine phosphorylation of IL-2Rbeta, JAK3, and STAT5, and abolished STAT5 DNA binding activity. Serine/threonine phosphorylation of IL-2Rbeta by a staurosporine-sensitive kinase also blocked its association with JAK3 and IL-2Rgamma in YT cells. Taken together, these data indicate that serine/threonine phosphorylation negatively regulates IL-2 signaling at multiple levels, including receptor complex formation and JAK3/STAT5 activation, and that this regulation is counteracted by PP2A. These findings also suggest that PP2A may serve as a therapeutic target for modulating JAK3/STAT5 activation in human disease.

  15. Postsynaptic Adenosine A2A Receptors Modulate Intrinsic Excitability of Pyramidal Cells in the Rat Basolateral Amygdala

    PubMed Central

    Rau, Andrew R.; Ariwodola, Olusegun J.

    2015-01-01

    Background: The basolateral amygdala plays a critical role in the etiology of anxiety disorders and addiction. Pyramidal neurons, the primary output cells of this region, display increased firing following exposure to stressors, and it is thought that this increase in excitability contributes to stress responsivity and the expression of anxiety-like behaviors. However, much remains unknown about the underlying mechanisms that regulate the intrinsic excitability of basolateral amygdala pyramidal neurons. Methods: Ex vivo gramicidin perforated patch recordings were conducted in current clamp mode where hyper- and depolarizing current steps were applied to basolateral amygdala pyramidal neurons to assess the effects of adenosine A2A receptor modulation on intrinsic excitability. Results: Activation of adenosine A2A receptors with the selective A2A receptor agonist CGS-21680 significantly increased the firing rate of basolateral amygdala pyramidal neurons in rat amygdala brain slices, likely via inhibition of the slow afterhyperpolarization potential. Both of these A2A receptor-mediated effects were blocked by preapplication of a selective A2A receptor antagonist (ZM-241385) or by intra-pipette infusion of a protein kinase A inhibitor, suggesting a postsynaptic locus of A2A receptors on basolateral amygdala pyramidal neurons. Interestingly, bath application of the A2A receptor antagonist alone significantly attenuated basolateral amygdala pyramidal cell firing, consistent with a role for tonic adenosine in the regulation of the intrinsic excitability of these neurons. Conclusions: Collectively, these data suggest that adenosine, via activation of A2A receptors, may directly facilitate basolateral amygdala pyramidal cell output, providing a possible balance for the recently described inhibitory effects of adenosine A1 receptor activation on glutamatergic excitation of basolateral amygdala pyramidal cells. PMID:25716780

  16. Behavioural and neurochemical characterization of the adenosine A2A receptor antagonist ST1535.

    PubMed

    Galluzzo, Mariangela; Pintor, Anita; Pèzzola, Antonella; Grieco, Rosa; Borsini, Franco; Popoli, Patrizia

    2008-01-28

    ST1535 (2-butyl-9-methyl-8-(2H-1,2,3-triazol 2-yl)-9 H-purin-6-ylamine) is a novel compound showing a preferential adenosine A(2A) receptor antagonist profile. To explore the potential neuroprotective profile of this compound, we evaluated whether ST1535 prevented quinolinic acid (QA)-induced glutamate outflow in the rat striatum (a reliable index of neuroprotective activity in vivo). Microdialysis experiments were performed in naive Wistar rats. In these experiments, a behaviourally active and inactive doses of ST1535 were used. Both doses significantly prevented QA-induced glutamate outflow in the striatum. These results show that ST1535 protects towards striatal excitotoxicity, even though its reduced A(2A)/A(1) selectivity might limit its actual neuroprotective potential.

  17. In vitro metabolism studies of new adenosine A 2A receptor antagonists.

    PubMed

    Marucci, Gabriella; Finaurini, Sara; Buccioni, Michela; Lammi, Carmen; Kandhavelu, Meenakshisundaram; Volpini, Rosaria; Ricciutelli, Massimo; Angeli, Piero; Commandeur, Jan N M; Cristalli, Gloria

    2008-12-01

    Evidence, obtained in rodent and primate models of Parkinson's disease (PD) and in preliminary clinical trials, indicates that adenosine A(2A) receptor antagonists might represent a promising non-dopaminergic therapeutic tool for the treatment of PD. Recently, we have reported the biological evaluation of 8-substituted 9-ethyladenines (ANR) as new A(2A) receptor antagonists, three of which (ANR 82, ANR 94, and ANR 152) showed high efficacy in in vivo models for Parkinson's. Understanding the metabolic pathways of new drug candidates is an important aspect of drug discovery. The ANR compounds have been investigated in order to clarify their activity on rat liver microsomes, and more specifically on recombinant human cytochrome P450 2D6 (CYP2D6). The metabolites of all three compounds were detected by liquid chromatography/tandem mass spectrometry (LC-MS/MS). The results indicate that this class of 9-ethyladenines is metabolized only to a fraction of 1.5-5%. These compounds also act as potent mechanism-based inhibitors of CYP450 and in particular of human isoform CYP2D6. Kinetic-analysis of enzyme inactivation was used to describe the effect of these time-dependent inhibitors and to derive the inhibition parameters K(inact) and K(i) defined with respect to the O-demethylation of dextromethorphan.

  18. A homology-based model of the human 5-HT2A receptor derived from an in silico activated G-protein coupled receptor

    NASA Astrophysics Data System (ADS)

    Chambers, James J.; Nichols, David E.

    2002-07-01

    A homology-based model of the 5-HT2A receptor was produced utilizing an activated form of the bovine rhodopsin (Rh) crystal structure [1,2]. In silico activation of the Rh structure was accomplished by isomerization of the 11- cis-retinal (1) chromophore, followed by constrained molecular dynamics to relax the resultant high energy structure. The activated form of Rh was then used as a structural template for development of a human 5-HT2A receptor model. Both the 5-HT2A receptor and Rh are members of the G-protein coupled receptor (GPCR) super-family. The resulting homology model of the receptor was then used for docking studies of compounds representing a cross-section of structural classes that activate the 5-HT2A receptor, including ergolines, tryptamines, and amphetamines. The ligand/receptor complexes that ensued were refined and the final binding orientations were observed to be compatible with much of the data acquired through both diversified ligand design and site directed mutagenesis.

  19. Past, present and future of A(2A) adenosine receptor antagonists in the therapy of Parkinson's disease.

    PubMed

    Armentero, Marie Therese; Pinna, Annalisa; Ferré, Sergi; Lanciego, José Luis; Müller, Christa E; Franco, Rafael

    2011-12-01

    Several selective antagonists for adenosine A(2A) receptors (A(2A)R) are currently under evaluation in clinical trials (phases I to III) to treat Parkinson's disease, and they will probably soon reach the market. The usefulness of these antagonists has been deduced from studies demonstrating functional interactions between dopamine D₂ and adenosine A(2A) receptors in the basal ganglia. At present it is believed that A(2A)R antagonists can be used in combination with the dopamine precursor L-DOPA to minimize the motor symptoms of Parkinson's patients. However, a considerable body of data indicates that in addition to ameliorating motor symptoms, adenosine A(2A)R antagonists may also prevent neurodegeneration. Despite these promising indications, one further issue must be considered in order to develop fully optimized antiparkinsonian drug therapy, namely the existence of (hetero)dimers/oligomers of G protein-coupled receptors, a topic that is currently the focus of intense debate within the scientific community. Dopamine D₂ receptors (D₂Rs) expressed in the striatum are known to form heteromers with A(2A) adenosine receptors. Thus, the development of heteromer-specific A(2A) receptor antagonists represents a promising strategy for the identification of more selective and safer drugs.

  20. Agonist properties of N,N-dimethyltryptamine at serotonin 5-HT2A and 5-HT2C receptors.

    PubMed

    Smith, R L; Canton, H; Barrett, R J; Sanders-Bush, E

    1998-11-01

    Extensive behavioral and biochemical evidence suggests an agonist role at the 5-HT2A receptor, and perhaps the 5-HT2C receptor, in the mechanism of action of hallucinogenic drugs. However the published in vitro pharmacological properties of N,N-dimethyltryptamine (DMT), an hallucinogenic tryptamine analog, are not consistent with this hypothesis. We, therefore, undertook an extensive investigation into the properties of DMT at 5-HT2A and 5-HT2C receptors. In fibroblasts transfected with the 5-HT2A receptor or the 5-HT2C receptor, DMT activated the major intracellular signaling pathway (phosphoinositide hydrolysis) to an extent comparable to that produced by serotonin. Because drug efficacy changes with receptor density and cellular microenvironment, we also examined the properties of DMT in native preparations using a behavioral and biochemical approach. Rats were trained to discriminate an antagonist ketanserin from an agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in a two-lever choice paradigm. Pharmacological studies showed that responding on the DOI and ketanserin lever reflected agonist and antagonist activity at 5-HT2A receptors, and hence, was a suitable model for evaluating the in vivo functional properties of DMT. Like other 5-HT2A receptor agonists, DMT substituted fully for DOI. Intact choroid plexus was used to evaluate the agonist properties at endogenous 5-HT2C receptors; DMT was a partial agonist at 5-HT2C receptors in this native preparation. Thus, we conclude that DMT behaves as an agonist at both 5-HT2A and 5-HT2A receptors. One difference was evident in that the 5-HT2C, but not the 5-HT2A, receptor showed a profound desensitization to DMT over time. This difference is interesting in light of the recent report that the hallucinogenic activity of DMT does not tolerate in humans and suggests the 5-HT2C receptor plays a less prominent role in the action of DMT.

  1. Age-dependent effects of the 5-hydroxytryptamine-2a-receptor polymorphism (His452Tyr) on human memory.

    PubMed

    Papassotiropoulos, Andreas; Henke, Katharina; Aerni, Amanda; Coluccia, Daniel; Garcia, Esmeralda; Wollmer, Marc A; Huynh, Kim-Dung; Monsch, Andreas U; Stähelin, Hannes B; Hock, Christoph; Nitsch, Roger M; de Quervain, Dominique J-F

    2005-05-31

    A polymorphism (His452Tyr) of the 5-hydroxytryptamine (5-HT)2a receptor is associated with episodic memory in healthy young humans. Because 5-HT2a-receptor density decreases with increasing age, we tested whether the 5-HT2a receptor genotype effect on memory is influenced by age. We investigated the association of the His452Tyr genotype with memory performance in 622 healthy study participants aged from 18 to 90 years. In young to middle-aged participants, age significantly influenced genotype effects on episodic memory: the His452Tyr genotype exerted a significant influence on memory only in young participants. In the group of elderly cognitively healthy participants, the His452Tyr genotype did not affect memory performance. We conclude that age strongly modulates the effect of the 5-HT2a receptor polymorphism at residue 452 on episodic memory.

  2. A1 and A2a receptors mediate inhibitory effects of adenosine on the motor activity of human colon.

    PubMed

    Fornai, M; Antonioli, L; Colucci, R; Ghisu, N; Buccianti, P; Marioni, A; Chiarugi, M; Tuccori, M; Blandizzi, C; Del Tacca, M

    2009-04-01

    Experimental evidence in animal models suggests that adenosine is involved in the regulation of digestive functions. This study examines the influence of adenosine on the contractile activity of human colon. Reverse transcription-polymerase chain reaction revealed A(1) and A(2a) receptor expression in colonic neuromuscular layers. Circular muscle preparations were connected to isotonic transducers to determine the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A(1) receptor antagonist), ZM 241385 (A(2a) receptor antagonist), CCPA (A(1) receptor agonist) and 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamide-adenosine (CGS 21680; A(2a) receptor agonist) on motor responses evoked by electrical stimulation or carbachol. Electrically evoked contractions were enhanced by DPCPX and ZM 241385, and reduced by CCPA and CGS 21680. Similar effects were observed when colonic preparations were incubated with guanethidine (noradrenergic blocker), L-732,138, GR-159897 and SB-218795 (NK receptor antagonists). However, in the presence of guanethidine, NK receptor antagonists and N(omega)-propyl-L-arginine (NPA; neuronal nitric oxide synthase inhibitor), the effects of DPCPX and CCPA were still evident, while those of ZM 241385 and CGS 21680 no longer occurred. Carbachol-induced contractions were unaffected by A(2a) receptor ligands, but they were enhanced or reduced by DPCPX and CCPA, respectively. When colonic preparations were incubated with guanethidine, NK antagonists and atropine, electrically induced relaxations were partly reduced by ZM 241385 or NPA, but unaffected by DPCPX. Dipyridamole or application of exogenous adenosine reduced electrically and carbachol-evoked contractions, whereas adenosine deaminase enhanced such motor responses. In conclusion, adenosine exerts an inhibitory control on human colonic motility. A(1) receptors mediate direct modulating actions on smooth muscle, whereas A(2a) receptors operate through inhibitory nitrergic nerve pathways.

  3. Influence of CGS 21680, a selective adenosine A(2A) receptor agonist, on NMDA receptor function and expression in the brain of Huntington's disease mice.

    PubMed

    Ferrante, Antonella; Martire, Alberto; Armida, Monica; Chiodi, Valentina; Pézzola, Antonella; Potenza, Rosa Luisa; Domenici, Maria Rosaria; Popoli, Patrizia

    2010-04-06

    The effect of chronic treatment with the selective adenosine A(2A) receptor agonist CGS 21680 on N-Methyl-d-Aspartate (NMDA) receptor function and expression has been studied in the striatum and cortex of R6/2 mice, a genetic mouse model of Huntington's disease (HD). Starting from 8weeks of age, R6/2 and wild type (WT) mice were treated daily with CGS 21680 (0.5mg/kg i.p.) for 3weeks and the expression levels of NMDA receptor subunits were then evaluated. In addition, to study CGS 21680-induced changes in NMDA receptor function, NMDA-induced toxicity in corticostriatal slices from both R6/2 and WT mice was investigated. We found that CGS 21680 increased NR2A subunit expression and the NR2A/NR2B ratio in the cortex of R6/2 mice, having no effect in WT mice. In the striatum, CGS 21680 reduced NR1 expression in both R6/2 and WT mice while the effect on NR2A and NR2/NR2B expression was genotype-dependent, reducing and increasing their expression in WT and R6/2 mice, respectively. On the contrary, NMDA-induced toxicity in corticostriatal slices was not modified by the treatment in WT or HD mice. These results demonstrate that in vivo activation of A(2A) receptors modulates the subunit composition of NMDA receptors in the brain of HD mice.

  4. Serotonin-2C and -2A Receptor Co-expression on Cells in the Rat Medial Prefrontal Cortex

    PubMed Central

    Nocjar, Christine; Alex, Katherine D; Sonneborn, Alex; Abbas, Atheir I; Roth, Bryan L; Pehek, Elizabeth A

    2015-01-01

    Neural function within the medial prefrontal cortex (mPFC) regulates normal cognition, attention and impulse control, implicating neuroregulatory abnormalities within this region in mental dysfunction related to schizophrenia, depression and drug abuse. Both serotonin -2A (5-HT2A) and -2C (5-HT2C) receptors are known to be important in neuropsychiatric drug action and are distributed throughout the mPFC. However, their interactive role in serotonergic cortical regulation is poorly understood. While the main signal transduction mechanism for both receptors is stimulation of phosphoinositide production, they can have opposite effects downstream. 5-HT2A versus 5-HT2C receptor activation oppositely regulates behavior and can oppositely affect neurochemical release within the mPFC. These distinct receptor effects could be caused by their differential cellular distribution within the cortex and/or other areas. It is known that both receptors are located on GABAergic and pyramidal cells within the mPFC, but it is not clear whether they are expressed on the same or different cells. The present work employed immunofluorescence with confocal microscopy to examine this in layers V-VI of the prelimbic mPFC. The majority of GABA cells in the deep prelimbic mPFC expressed 5-HT2C receptor immunoreactivity. Furthermore, most cells expressing 5-HT2C receptor immunoreactivity notably co-expressed 5-HT2A receptors. However, 27% of 5-HT2C receptor immunoreactive cells were not GABAergic, indicating that a population of prelimbic pyramidal projection cells could express the 5-HT2C receptor. Indeed, some cells with 5-HT2C and 5-HT2A receptor co-labeling had a pyramidal shape and were expressed in the typical layered fashion of pyramidal cells. This indirectly demonstrates that 5-HT2C and 5-HT2A receptors may be commonly co-expressed on GABAergic cells within the deep layers of the prelimbic mPFC and perhaps co-localized on a small population of local pyramidal projection cells. Thus a

  5. Characterization of [125I]ZM 241385 binding to adenosine A2A receptors in the pineal of sheep brain.

    PubMed

    Yan, X; Koos, B J; Kruger, L; Linden, J; Murray, T F

    2006-06-22

    Adenosine is a ubiquitous neuromodulator and homeostatic regulator that exerts its physiologic actions through activation of A(1), A(2A), A(2B) and A(3) adenosine receptor subtypes. In the central nervous system, adenosine's action in neurons is manifested in its modulation of tonic inhibitory control. Adenosine released in the brain during hypoxia has critical depressant effects on breathing in fetal and newborn mammals, an action suggested to be mediated by A(2A) receptors in the posteromedial thalamus. In an effort to more accurately define the spatial distribution of adenosine A(2A) receptors in fetal sheep diencephalon, we have used a receptor autoradiographic technique utilizing an iodinated radioligand [(125)I]ZM 241385, which has greater sensitivity and resolution than the tritiated compound. The distribution of ligand binding sites in the fetal sheep diencephalon indicated that the highest levels of binding were in select thalamic nuclei, including those implicated in hypoxic depression of fetal breathing, and the pineal. Given the high density of labeled A(2A) receptors in the pineal, these sites were characterized more fully in homogenate radioligand binding assays. These data indicate that [(125)I]ZM 241385 binding sites display a pharmacological signature consistent with that of adenosine A(2A) receptors and are expressed at similar levels in fetal, lamb and adult ovine brain. The adenosine A(2A) receptor pharmacologic signature of the [(125)I]ZM 241385 binding site in pineal cell membranes generalized to the site characterized in membranes derived from other portions of the lamb thalamus, including the sector involved in hypoxic inhibition of fetal breathing. These results have important implications for the functional roles of adenosine A(2A) receptors in the thalamus and pineal of sheep brain.

  6. Enhancement of AMPA currents and GluR1 membrane expression through PKA-coupled adenosine A(2A) receptors.

    PubMed

    Dias, Raquel B; Ribeiro, Joaquim A; Sebastião, Ana M

    2012-02-01

    Phosphorylation of glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by Protein Kinase A (PKA) is known to regulate AMPA receptor (AMPAR) trafficking and stabilization at the postsynaptic membrane, which in turn is one of the key mechanisms by which synaptic transmission and plasticity are tuned. However, not much is known as to how Gs-coupled receptors contribute to endogenous PKA-mediated regulation of AMPA receptor function. Here we report that activation of the excitatory A(2A) adenosine receptor by 2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680, 1-30 nM) facilitates AMPA-evoked currents in CA1 pyramidal neurons, by a mechanism dependent on PKA activation, but not on protein synthesis. This modulation of AMPA currents was mimicked by forskolin (1 μM) and did not occur in stratum radiatum interneurons. Superfusion of the A(2A) receptor agonist also caused an increase in the amplitude of miniature excitatory postsynaptic currents (mEPSCs), as well as in the membrane levels of GluR1 subunits phosphorylated at the PKA site (Ser845). The impact of this increase on GluR1-containing AMPA receptor expression was evidenced by the potentiation of LTP at the CA3-CA1 synapse that followed brief activation of A(2A) receptors. We thus propose that in conditions of increased adenosine availability, A(2A) receptor activation is responsible for setting part of the endogenous GluR1 Ser-845 phosphorylation tonus and hence, the availability of the GluR1-containing AMPA receptor extrasynaptic pool for synaptic insertion and reinforcement of synaptic strength.

  7. Selective adenosine A2A receptor agonists and antagonists protect against spinal cord injury through peripheral and central effects

    PubMed Central

    2011-01-01

    Background Permanent functional deficits following spinal cord injury (SCI) arise both from mechanical injury and from secondary tissue reactions involving inflammation. Enhanced release of adenosine and glutamate soon after SCI represents a component in the sequelae that may be responsible for resulting functional deficits. The role of adenosine A2A receptor in central ischemia/trauma is still to be elucidated. In our previous studies we have demonstrated that the adenosine A2A receptor-selective agonist CGS21680, systemically administered after SCI, protects from tissue damage, locomotor dysfunction and different inflammatory readouts. In this work we studied the effect of the adenosine A2A receptor antagonist SCH58261, systemically administered after SCI, on the same parameters. We investigated the hypothesis that the main action mechanism of agonists and antagonists is at peripheral or central sites. Methods Spinal trauma was induced by extradural compression of SC exposed via a four-level T5-T8 laminectomy in mouse. Three drug-dosing protocols were utilized: a short-term systemic administration by intraperitoneal injection, a chronic administration via osmotic minipump, and direct injection into the spinal cord. Results SCH58261, systemically administered (0.01 mg/kg intraperitoneal. 1, 6 and 10 hours after SCI), reduced demyelination and levels of TNF-α, Fas-L, PAR, Bax expression and activation of JNK mitogen-activated protein kinase (MAPK) 24 hours after SCI. Chronic SCH58261 administration, by mini-osmotic pump delivery for 10 days, improved the neurological deficit up to 10 days after SCI. Adenosine A2A receptors are physiologically expressed in the spinal cord by astrocytes, microglia and oligodendrocytes. Soon after SCI (24 hours), these receptors showed enhanced expression in neurons. Both the A2A agonist and antagonist, administered intraperitoneally, reduced expression of the A2A receptor, ruling out the possibility that the neuroprotective effects

  8. New adenosine A2A receptor antagonists: actions on Parkinson's disease models.

    PubMed

    Pinna, Annalisa; Volpini, Rosaria; Cristalli, Gloria; Morelli, Micaela

    2005-04-11

    The 8-substituted 9-ethyladenine derivatives: 8-bromo-9-ethyladenine (ANR 82), 8-ethoxy- 9-ethyladenine (ANR 94), and 8-furyl-9-ethyladenine (ANR 152) have been characterized in vitro as adenosine receptor antagonists. Adenosine is deeply involved in the control of motor behaviour and substantial evidences indicate that adenosine A(2A) receptor antagonists improve motor deficits in animal models of Parkinson's disease. On this basis, the efficacy of ANR 82, ANR 94, and ANR 152 in rat models of Parkinson's disease was evaluated. All compounds tested reversed the catalepsy induced by haloperidol. However, in unilaterally 6-hydroxydopamine-lesioned rats, only ANR 94 and ANR 152 potentiated l-dihydroxy-phenylalanine (l-DOPA) effect on turning behaviour and induced contralateral turning behaviour in rats sensitised to l-DOPA. Taken together the results of this study indicate that some 8-substituted 9-ethyladenine derivatives ameliorate motor deficits in rat models of Parkinson's disease, suggesting a potential therapeutic role of these compounds.

  9. A2A adenosine receptors are up-regulated in lymphocytes from amyotrophic lateral sclerosis patients.

    PubMed

    Vincenzi, Fabrizio; Corciulo, Carmen; Targa, Martina; Casetta, Ilaria; Gentile, Mauro; Granieri, Enrico; Borea, Pier Andrea; Popoli, Patrizia; Varani, Katia

    2013-09-01

    Adenosine, a purine nucleoside interacting with A1, A2A, A2B and A3 adenosine receptors (ARs), is a potent endogenous modulator of inflammatory and neuronal processes involved in the pathophysiology of several neurodegenerative diseases. In the present study, ARs were investigated in lymphocytes from patients with amyotrophic lateral sclerosis (ALS) and compared with age-matched healthy subjects. In ALS patients A2AARs were analysed by using RT-PCR, Western blotting and saturation binding experiments. The effect of A2AAR stimulation on cyclic AMP levels was evaluated in lymphocytes from ALS patients and healthy subjects. An up-regulation of A2AARs was observed in ALS patients with respect to healthy subjects while A1, A2B and A3AR affinity and density did not change. In ALS patients, the A2AAR density values correlated with the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) scores. Furthermore, the stimulation of A2AARs mediated a significant increase in cyclic AMP levels in lymphocytes from ALS patients, with a higher potency than in lymphocytes from healthy subjects. In conclusion, the positive correlation between A2AAR density and ALSFRS-R scores could indicate a possible protective effect of this receptor subtype, representing an interesting starting point for the study of alternative therapeutic approaches for ALS based on A2AAR modulation.

  10. Characterization of the promoter of human CRTh2, a prostaglandin D{sub 2} receptor

    SciTech Connect

    Quapp, Russell; Madsen, Norman; Cameron, Lisa

    2007-11-30

    Chemoattractant-receptor homologous molecule expressed on Th2 cells (CRTh2) is a receptor for prostaglandin (PG)D{sub 2}, a lipid mediator involved in allergic inflammation. CRTh2 is expressed by Th2 cells, eosinophils and basophils and PDG{sub 2}-CRTh2 signaling induces calcium mobilization, cell migration and expression of the Th2 cytokines IL-4, IL-5, and IL-13. Despite the role of CRTh2 in allergic inflammation, transcriptional regulation of this gene has not been studied. Here, we demonstrated that a reporter construct of the CRTh2 promoter was induced following T cell stimulation. This activity could be further enhanced by over-expression of GATA-3, but not NFAT2 or STAT6. Electromobility shift assay demonstrated GATA-3 binding to a probe from the CRTh2 promoter. This study provides the first detailed analysis of transcriptional regulation of the human CRTh2 promoter. These findings may help identify strategies to attenuate expression of this gene and influence the maintenance and proliferation of Th2 cells in allergic inflammation.

  11. A tail of two signals: the C terminus of the A(2A)-adenosine receptor recruits alternative signaling pathways.

    PubMed

    Gsandtner, Ingrid; Freissmuth, Michael

    2006-08-01

    G protein-coupled receptors are endowed with carboxyl termini that vary greatly in length and sequence. In most instances, the distal portion of the C terminus is dispensable for G protein coupling. This is also true for the A(2A)-adenosine receptor, where the last 100 amino acids are of very modest relevance to G(s) coupling. The C terminus was originally viewed mainly as the docking site for regulatory proteins of the beta-arrestin family. These beta-arrestins bind to residues that have been phosphorylated by specialized kinases (G protein-coupled receptor kinases) and thereby initiate receptor desensitization and endocytosis. More recently, it has become clear that many additional "accessory" proteins bind to C termini of G protein-coupled receptors. The article by Sun et al. in the current issue of Molecular Pharmacology identifies translin-associated protein-X as yet another interaction partner of the A(2A) receptor; translin-associated protein allows the A(2A) receptor to impinge on the signaling mechanisms by which p53 regulates neuronal differentiation, but the underlying signaling pathways are uncharted territory. With a list of five known interaction partners, the C terminus of the A(2A) receptor becomes a crowded place. Hence, there must be rules that regulate the interaction. This allows the C terminus to act as coincidence detector and as signal integrator. Despite our ignorance about the precise mechanisms, the article has exciting implications: the gene encoding for translin-associated protein-X maps to a locus implicated in some forms of schizophrenia; A(2A) receptor agonists are candidate drugs for the treatment of schizophrenic symptoms. It is of obvious interest to explore a possible link.

  12. Biochemical profile of YM992, a novel selective serotonin reuptake inhibitor with 5-HT2A receptor antagonistic activity.

    PubMed

    Hatanaka, K; Nomura, T; Hidaka, K; Takeuchi, H; Yatsugi, S; Fujii, M; Yamaguchi, T

    1996-01-01

    YM992, (S)-2-[[(7-fluoro-4-indanyl)oxy]methyl]morpholine monohydrochloride, exhibited the biochemical profile of a selective serotonin (5-HT) reuptake inhibitor (SSRI) with 5-HT2A receptor antagonistic activity. YM922 showed the same high affinity as fluoxetine against the 5-HT reuptake site (Ki = 21 nM) and a similar affinity to that of crazodone against the 5-HT2A receptor (Ki = 86 nM). In other receptor binding studies, an affinity for the adrenergic alpha 1 receptor (Ki = 200 nM) and 5-HT2C receptor (Ki = 680 nM) was observed. In a monoamine uptake study, YM992 showed a selective 5-HT uptake inhibition (IC50 = 0.15 microM), but only very weakly inhibited both noradrenaline (NA) and dopamine (DA) uptake (IC50 = 3.1 microM (NA), > 10 microM (DA)). YM992 was also found to potently inhibit the aggregation of human platelets (IC50 = 1.9 microM), revealing antagonistic activity for the 5-HT2A receptor in vitro. Enhanced serotonergic neurotransmission, in particular that mediated by the 5-HT1A receptor, has recently been reported to be important in the long-term treatment of depressive disorders with antidepressants. In addition, some 5-HT1A receptor-mediated responses are known to be potentiated by co-administration of 5-HT2A receptor antagonists. Thus, YM992, having both selective 5-HT reuptake inhibition and 5-HT2A antagonistic activity, might show potent therapeutic activity as a novel antidepressant in comparison with conventional SSRIs.

  13. Molecular dynamics of 5-HT1A and 5-HT2A serotonin receptors with methylated buspirone analogues

    NASA Astrophysics Data System (ADS)

    Bronowska, Agnieszka; Chilmonczyk, Zdzisław; Leś, Andrzej; Edvardsen, Øyvind; Østensen, Roy; Sylte, Ingebrigt

    2001-11-01

    In the present study experimentally determined ligand selectivity of three methylated buspirone analogues (denoted as MM2, MM5 and P55) towards 5-HT1A and 5-HT2A serotonin receptors was theoretically investigated on a molecular level. The relationships between the ligand structure and 5-HT1A and 5-HT2A receptor affinities were studied and the results were found to be in agreement with the available site-directed mutagenesis and binding affinity data. Molecular dynamics (MD) simulations of ligand-receptor complexes were performed for each investigated analogue, docked twice into the central cavity of 5-HT1A/5-HT2A, each time in a different orientation. Present results were compared with our previous theoretical results, obtained for buspirone and its non-methylated analogues. It was found that due to the presence of the methyl group in the piperazine ring the ligand position alters and the structure of the ligand-receptor complex is modified. Further, the positions of derivatives with pyrimidinyl aromatic moiety and quinolinyl moiety are significantly different at the 5-HT2A receptor. Thus, methylation of such derivatives alters the 3D structures of ligand-receptor complexes in different ways. The ligand-induced changes of the receptor structures were also analysed. The obtained results suggest, that helical domains of both receptors have different dynamical behaviour. Moreover, both location and topography of putative binding sites for buspirone analogues are different at 5-HT1A and 5-HT2A receptors.

  14. 5-HT2A receptor antagonist M100907 reduces serotonin synthesis: An autoradiographic study

    PubMed Central

    Hasegawa, Shu; Fikre-Merid, Maraki; Diksic, Mirko

    2013-01-01

    The effects of the administration of the serotonin (5-HT)2A antagonist, M100907, on 5-HT synthesis rates, were evaluated using the α-[14C]methyl-L-tryptophan (α-MTrp) autoradiographic method. In the treatment study, M100907 (10 mg/kg) was injected intraperitoneally 30 min before the α-MTrp injection (30 μCi over 2 min). A single dose of M100907 caused a significant decrease in the synthesis in the anterior olfactory nucleus, accumbens nucleus, frontal cortex, sensory-motor cortex, cingulate cortex, medial caudate-putamen, dorsal thalamus, substantia nigra, inferior collicus, raphe magnus nucleus, superior olive, and raphe pallidus nucleus. These data suggest that the terminal 5-HT2A receptors are involved in the regulation of 5-HT synthesis in the entire brain. Further, 5-HT synthesis is likely regulated by the 5-HT2A antagonistic property of M100907 in the cortices, anterior olfactory nucleus, caudate putamen, and nucleus accumbens. PMID:22056993

  15. Chronic methamphetamine treatment induces oxytocin receptor up-regulation in the amygdala and hypothalamus via an adenosine A2A receptor-independent mechanism.

    PubMed

    Zanos, Panos; Wright, Sherie R; Georgiou, Polymnia; Yoo, Ji Hoon; Ledent, Catherine; Hourani, Susanna M; Kitchen, Ian; Winsky-Sommerer, Raphaelle; Bailey, Alexis

    2014-04-01

    There is mounting evidence that the neuropeptide oxytocin is a possible candidate for the treatment of drug addiction. Oxytocin was shown to reduce methamphetamine self-administration, conditioned place-preference, hyperactivity and reinstatement in rodents, highlighting its potential for the management of methamphetamine addiction. Thus, we hypothesised that the central endogenous oxytocinergic system is dysregulated following chronic methamphetamine administration. We tested this hypothesis by examining the effect of chronic methamphetamine administration on oxytocin receptor density in mice brains with the use of quantitative receptor autoradiographic binding. Saline (4ml/kg/day, i.p.) or methamphetamine (1mg/kg/day, i.p.) was administered daily for 10 days to male, CD1 mice. Quantitative autoradiographic mapping of oxytocin receptors was carried out with the use of [(125)I]-vasotocin in brain sections of these animals. Chronic methamphetamine administration induced a region specific upregulation of oxytocin receptor density in the amygdala and hypothalamus, but not in the nucleus accumbens and caudate putamen. As there is evidence suggesting an involvement of central adenosine A2A receptors on central endogenous oxytocinergic function, we investigated whether these methamphetamine-induced oxytocinergic neuroadaptations are mediated via an A2A receptor-dependent mechanism. To test this hypothesis, autoradiographic oxytocin receptor binding was carried out in brain sections of male CD1 mice lacking A2A receptors which were chronically treated with methamphetamine (1mg/kg/day, i.p. for 10 days) or saline. Similar to wild-type animals, chronic methamphetamine administration induced a region-specific upregulation of oxytocin receptor binding in the amygdala and hypothalamus of A2A receptor knockout mice and no genotype effect was observed. These results indicate that chronic methamphetamine use can induce profound neuroadaptations of the oxytocinergic receptor

  16. Adenosine A(2A)-cannabinoid CB(1) receptor interaction: an integrative mechanism in striatal glutamatergic neurotransmission.

    PubMed

    Tebano, Maria Teresa; Martire, Alberto; Popoli, Patrizia

    2012-10-02

    The striatum is a subcortical area involved in sensorimotor, cognitive and emotional processes. Adenosine A(2A) receptors (A(2A)Rs) are highly expressed in the striatum, and their ability to establish functional and molecular interactions with many other receptors attributes to a pivotal role in the modulation and integration of striatal neurotransmission. This review will focus on the interaction between A(2A)Rs and cannabinoid CB(1) receptors (CB(1)Rs), taking it as a paradigmatic example of synaptic integration. Indeed, A(2A)Rs can exert an opposite (permissive vs. inhibitory) influence on CB1-dependent synaptic effect. These apparently irreconcilable functions could depend on a different role of pre- vs. postsynaptic A(2A)Rs, on their interaction with other receptors (namely adenosine A(1), metabotropic glutamate 5 and dopamine D2 receptors), and on whether A(2A)Rs form or not heteromers with CB(1)Rs. Besides providing a good example of the intricate pattern of events taking place in striatal synapses, the A(2A)/CB(1)R interaction proves very informative to understand the physiology of the basal ganglia and the mechanisms of related diseases. This article is part of a Special Issue entitled: Brain Integration.

  17. Serotonin 2A Receptor Gene Polymorphism in Korean Children with Attention-Deficit/Hyperactivity Disorder

    PubMed Central

    Cho, Soo-Churl; Kim, Boong-Nyun; Kim, Jae-Won; Yoo, Hee-Jeong; Hwang, Jun-Won; Cho, Dae-Yeon; Chung, Un-Sun; Park, Tae-Won

    2012-01-01

    Objective The purpose of this study was to investigate the association between the T102C polymorphism in the serotonin 2A receptor gene and attention-deficit/hyperactivity disorder (ADHD) in Korean patients. Methods A total of 189 Korean children with ADHD as well as both parents of the ADHD children and 150 normal children participated in this study. DNA was extracted from blood samples from all of the subjects, and genotyping was conducted. Based on the allele and genotype information obtained, case-control analyses were performed to compare the ADHD and normal children, and Transmission disequilibrium tests (TDTs) were used for family-based association testing (number of trios=113). Finally, according to the significant finding which was showed in the case-control analyses, the results of behavioral characterastics and neuropsychological test were compared between ADHD children with and without the C allele. Results In the case-control analyses, statistically significant differences were detected in the frequencies of genotypes containing the C allele (χ2=4.73, p=0.030). In the family-based association study, TDTs failed to detect linkage disequilibrium of the T102C polymorphism associated with ADHD children. In the ADHD children, both the mean reaction time and the standard deviation of the reaction time in the auditory continuous performance test were longer in the group with the C allele compared to the group without the C allele. Conclusion The results of this study suggest that there is a significant genetic association between the T102C polymorphism in the serotonin 2A receptor gene and ADHD in Korean children. PMID:22993527

  18. Common α2A and α2C adrenergic receptor polymorphisms do not affect plasma membrane trafficking.

    PubMed

    Hurt, Carl M; Sorensen, Matt W; Angelotti, Timothy

    2014-06-01

    Various naturally occurring polymorphic forms of human G protein-coupled receptors (GPCRs) have been identified and linked to diverse pathological diseases, including receptors for vasopressin type 2 (nephrogenic diabetes insipidus) and gonadotropin releasing hormone (hypogonadotropic hypogonadism). In most cases, polymorphic amino acid mutations disrupt protein folding, altering receptor function as well as plasma membrane expression. Other pathological GPCR variants have been found that do not alter receptor function, but instead affect only plasma membrane trafficking (e.g., delta opiate and histamine type 1 receptors). Thus, altered membrane trafficking with retained receptor function may be another mechanism causing polymorphic GPCR dysfunction. Two common human α2A and α2C adrenergic receptor (AR) variants have been identified (α2A N251K and α2C Δ322-325 ARs), but pharmacological analysis of ligand binding and second messenger signaling has not consistently demonstrated altered receptor function. However, possible alterations in plasma membrane trafficking have not been investigated. We utilized a systematic approach previously developed for the study of GPCR trafficking motifs and accessory proteins to assess whether these α2 AR variants affected intracellular trafficking or plasma membrane expression. By combining immunofluorescent microscopy, glycosidic processing analysis, and quantitative fluorescent-activated cell sorting (FACS), we demonstrate that neither variant receptor had altered intracellular localization, glycosylation, nor plasma membrane expression compared to wild-type α2 ARs. Therefore, pathopharmacological properties of α2A N251K and α2C Δ322-325 ARs do not appear to be due to altered receptor pharmacology or plasma membrane trafficking, but may involve interactions with other intracellular signaling cascades or proteins.

  19. A2A adenosine receptor regulates the human blood brain barrier permeability

    PubMed Central

    Kim, Do-Geun; Bynoe, Margaret S.

    2015-01-01

    The blood brain barrier (BBB) symbolically represents the gateway to the central nervous system. It is a single layer of specialized endothelial cells that coats the central nervous system (CNS) vasculature and physically separates the brain environment from the blood constituents, to maintain the homeostasis of the CNS. However, this protective measure is a hindrance to the delivery of therapeutics to treat neurological diseases. Here, we show that activation of A2A adenosine receptor (AR) with an FDA-approved agonist potently permeabilizes an in vitro primary human brain endothelial barrier (hBBB) to the passage of chemotherapeutic drugs and T cells. T cell migration under AR signaling occurs primarily by paracellular transendothelial route. Permeabilization of the hBBB is rapid, time-dependent and reversible and is mediated by morphological changes in actin-cytoskeletal reorganization induced by RhoA signaling and a potent down-regulation of Claudin-5 and VE-Cadherin. Moreover, the kinetics of BBB permeability in mice closely overlaps with the permeability kinetics of the hBBB. These data suggest that activation of A2A AR is an endogenous mechanism that may be used for CNS drug delivery in human. PMID:25262373

  20. Adenosine A2A receptor blockade differentially influences excitotoxic mechanisms at pre- and postsynaptic sites in the rat striatum.

    PubMed

    Tebano, Maria Teresa; Pintor, Annita; Frank, Claudio; Domenici, Maria Rosaria; Martire, Alberto; Pepponi, Rita; Potenza, Rosa Luisa; Grieco, Rosa; Popoli, Patrizia

    2004-07-01

    Adenosine A(2A) receptor antagonists are being regarded as potential neuroprotective drugs, although the mechanisms underlying their effects need to be better studied. The aim of this work was to investigate further the mechanism of the neuroprotective action of A(2A) receptor antagonists in models of pre- and postsynaptic excitotoxicity. In microdialysis studies, the intrastriatal perfusion of the A(2A) receptor antagonist ZM 241385 (5 and 50 nM) significantly reduced, in an inversely dose-dependent way, the raise in glutamate outflow induced by 5 mM quinolinic acid (QA). In rat corticostriatal slices, ZM 241385 (30-100 nM) significantly reduced 4-aminopyridine (4-AP)-induced paired-pulse inhibition (PPI; an index of neurotransmitter release), whereas it worsened the depression of field potential amplitude elicited by N-methyl-D-aspartate (NMDA; 12.5 and 50 microM). The A(2A) antagonist SCH 58261 (30 nM) mimicked the effects of ZM 241385, whereas the A(2A) agonist CGS 21680 (100 nM) showed a protective influence toward 50 microM NMDA. In rat striatal neurons, 50 nM ZM 241385 did not affect the increase in [Ca(2+)](i) or the release of lactate dehydrogenase (LDH) induced by 100 and 300 microM NMDA, respectively. The ability of ZM 241385 to prevent QA-induced glutamate outflow and 4-AP-induced effects confirms that A(2A) receptor antagonists have inhibitory effects on neurotransmitter release, whereas the results obtained toward NMDA-induced effects suggest that A(2A) receptor blockade does not reduce, or even amplifies, excitotoxic mechanisms due to direct NMDA receptor stimulation. This indicates that the neuroprotective potential of A(2A) antagonists may be evident mainly in models of neurodegeneration in which presynaptic mechanisms play a major role.

  1. Age-dependent effects on social interaction of NMDA GluN2A receptor subtype-selective antagonism.

    PubMed

    Green, Torrian L; Burket, Jessica A; Deutsch, Stephen I

    2016-07-01

    NMDA receptor-mediated neurotransmission is implicated in the regulation of normal sociability in mice. The heterotetrameric NMDA receptor is composed of two obligatory GluN1 and either two "modulatory" GluN2A or GluN2B receptor subunits. GluN2A and GluN2B-containing receptors differ in terms of their developmental expression, distribution between synaptic and extrasynaptic locations, and channel kinetic properties, among other differences. Because age-dependent differences in disruptive effects of GluN2A and GluN2B subtype-selective antagonists on sociability and locomotor activity have been reported in rats, the current investigation explored age-dependent effects of PEAQX, a GluN2A subtype-selective antagonist, on sociability, stereotypic behaviors emerging during social interaction, and spatial working memory in 4- and 8-week old male Swiss Webster mice. The data implicate an age-dependent contribution of GluN2A-containing NMDA receptors to the regulation of normal social interaction in mice. Specifically, at a dose of PEAQX devoid of any effect on locomotor activity and mouse rotarod performance, the social interaction of 8-week old mice was disrupted without any effect on the social salience of a stimulus mouse. Moreover, PEAQX attenuated stereotypic behavior emerging during social interaction in 4- and 8-week old mice. However, PEAQX had no effect on spontaneous alternations, a measure of spatial working memory, suggesting that neural circuits mediating sociability and spatial working memory may be discrete and dissociable from each other. Also, the data suggest that the regulation of stereotypic behaviors and sociability may occur independently of each other. Because expression of GluN2A-containing NMDA receptors occurs at a later developmental stage, they may be more involved in mediating the pathogenesis of ASDs in patients with histories of "regression" after a period of normal development than GluN2B receptors.

  2. 5-HT2A receptors control body temperature in mice during LPS-induced inflammation via regulation of NO production.

    PubMed

    Voronova, Irina P; Khramova, Galina M; Kulikova, Elizabeth A; Petrovskii, Dmitrii V; Bazovkina, Daria V; Kulikov, Alexander V

    2016-01-01

    G protein-coupled 5-HT2A receptors are involved in the regulation of numerous normal and pathological physiological functions. At the same time, its involvement in the regulation of body temperature (Tb) in normal conditions is obscure. Here we study the effect of the 5-HT2A receptor activation or blockade on Tb in sick animals. The experiments were carried out on adult C57BL/6 mouse males. Systemic inflammation and sickness were produced by lipopolysaccharide (LPS, 0.1mg/kg, ip), while the 5-HT2A receptor was stimulated or blocked through the administration of the receptor agonist DOI or antagonist ketanserin (1mg/kg), respectively. LPS, DOI or ketanserin alone produced no effect on Tb. However, administration of LPS together with a peripheral or central ketanserin injection reduced Tb (32.2°C). Ketanserin reversed the LPS-induced expression of inducible NO synthase in the brain. Consequently, an involvement of NO in the mechanism of the hypothermic effect of ketanserin in sick mice was hypothesized. Administration of LPS together with NO synthase inhibitor, l-nitro-arginine methyl ester (60mg/kg, ip) resulted in deep (28.5°C) and prolonged (8h) hypothermia, while administration of l-nitro-arginine methyl ester alone produced no effect on Tb. Thus, 5-HT2A receptors play a key role in Tb control in sick mice. Blockade of this GPCR produces hypothermia in mice with systemic inflammation via attenuation of LPS-induced NO production. These results indicate an unexpected role of 5-HT2A receptors in inflammation and NO production and have a considerable biological impact on understanding the mechanism of animal adaptation to pathogens and parasites. Moreover, adverse side effects of 5-HT2A receptor antagonists in patients with inflammation may be expected.

  3. The selective 5-HT2A receptor antagonist M100907 enhances antidepressant-like behavioral effects of the SSRI fluoxetine.

    PubMed

    Marek, Gerard J; Martin-Ruiz, Raul; Abo, Allyson; Artigas, Francesc

    2005-12-01

    The addition of low doses of atypical antipsychotic drugs, which saturate 5-HT(2A) receptors, enhances the therapeutic effect of selective serotonin (5-hydroxytryptamine; 5-HT) reuptake inhibitors (SSRIs) in patients with major depression as well as treatment-refractory obsessive-compulsive disorder. The purpose of the present studies was to test the effects of combined treatment with a low dose of a highly selective 5-HT(2A) receptor antagonist (M100907; formerly MDL 100,907) and low doses of a SSRI using a behavioral screen in rodents (the differential-reinforcement-of low rate 72-s schedule of reinforcement; DRL 72-s) which previously has been shown to be sensitive both to 5-HT(2) antagonists and SSRIs. M100907 has a approximately 100-fold or greater selectivity at 5-HT(2A) receptors vs other 5-HT receptor subtypes, and would not be expected to appreciably occupy non-5-HT(2A) receptors at doses below 100 microg/kg. M100907 increased the reinforcement rate, decreased the response rate, and shifted the inter-response time distributions to the right in a pattern characteristic of antidepressant drugs. In addition, a positive synergistic interaction occurred when testing low doses of the 5-HT(2A) receptor antagonist (6.25-12.5 microg/kg) with clinically relevant doses of the SSRI fluoxetine (2.5-5 mg/kg), which both exerted minimal antidepressant-like effects by themselves. In vivo microdialysis study revealed that a low dose of M100907 (12.5 microg/kg) did not elevate extracellular 5-HT levels in the prefrontal cortex over those observed with fluoxetine alone (5 mg/kg). These results will be discussed in the context that the combined blockade of 5-HT(2A) receptors and serotonin transporters (SERT) may result in greater efficacy in treating neuropsychiatric syndromes than blocking either site alone.

  4. A2A receptor antagonists do not induce dyskinesias in drug-naive or L-dopa sensitized rats.

    PubMed

    Jones, N; Bleickardt, C; Mullins, D; Parker, E; Hodgson, R

    2013-09-01

    L-dopa, the precursor to dopamine, is currently the gold standard treatment for Parkinson's disease (PD). However, chronic exposure is associated with L-dopa-induced dyskinesias (LIDs), a serious side effect characterized by involuntary movements. Adenosine A2A receptor antagonists have been studied as a novel non-dopaminergic PD treatment. Because A2A receptor antagonists do not act on dopamine receptors, it has been hypothesized that they will not induce dyskinesias characteristic of L-dopa. To test this hypothesis in a rodent model, the A2A receptor antagonists SCH 412348 (3 mg/kg), vipadenant (10 mg/kg), caffeine (30 mg/kg), or istradefylline (3 mg/kg) were chronically (19-22 days) administered to Sprague Dawley rats, and dyskinetic behaviors were scored across this chronic dosing paradigm. Unlike L-dopa, there was no evidence of dyskinetic activity resulting from any of the four A2A receptor antagonists tested. When delivered to animals previously sensitized with L-dopa (6 mg/kg), SCH 412348, vipadenant, caffeine or istradefylline treatment produced no dyskinesias. When administered in combination with L-dopa (6 mg/kg), SCH 412348 (3 mg/kg) neither exacerbated nor prevented the induction of LIDs over the course of 19 days of treatment. Collectively, our data indicate that A2A receptor antagonists are likely to have a reduced dyskinetic liability relative to L-dopa but do not block dyskinesias when coadministered with L-dopa. Clinical studies are required to fully understand the dyskinesia profiles of A2A receptor antagonists.

  5. Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine.

    PubMed

    Doré, Andrew S; Robertson, Nathan; Errey, James C; Ng, Irene; Hollenstein, Kaspar; Tehan, Ben; Hurrell, Edward; Bennett, Kirstie; Congreve, Miles; Magnani, Francesca; Tate, Christopher G; Weir, Malcolm; Marshall, Fiona H

    2011-09-07

    Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A(2A) receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A(2A) selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins.

  6. Identification and characterization of a truncated variant of the 5-hydroxytryptamine(2A) receptor produced by alternative splicing.

    PubMed

    Guest, P C; Salim, K; Skynner, H A; George, S E; Bresnick, J N; McAllister, G

    2000-09-08

    We have identified an alternatively spliced 5-hydroxytryptamine 2A receptor (5-HT(2A)-R) transcript by PCR of human brain cDNA using degenerate oligonucleotide primers to transmembrane (TM) domains 3 and 7 of the 5-HT(2)-R subfamily. The variant contains a 118-bp insertion at the exon II/III boundary of the 5-HT(2A)-R, which produces a frame shift in the coding sequence and a premature stop codon. PCR analysis showed that the truncated receptor (5-HT(2A-tr)) and native 5-HT(2A)-R were co-expressed in most brain tissues, with the highest levels being found in hippocampus, corpus collosum, amygdala and caudate nucleus. Western blot analysis of HEK-293 cells transfected transiently with a 5-HT(2A-tr) construct showed that a 30-kDa protein was expressed on cell membranes. Co-transfection studies showed no effect of the 5-HT(2A-tr) variant on 3H-ketanserin binding to the native 5-HT(2A)-R or on functional coupling of the 5-HT(2A)-R to 5-HT-stimulated Ca(2+) mobilization. The functional significance of the 5-HT(2A-tr) variant and other truncated receptors remains to be established.

  7. Suppression of adenosine 2a receptor (A2aR)-mediated adenosine signaling improves disease phenotypes in a mouse model of amyotrophic lateral sclerosis.

    PubMed

    Ng, Seng Kah; Higashimori, Haruki; Tolman, Michaela; Yang, Yongjie

    2015-05-01

    Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease in which the majority of upper and lower motor neurons are degenerated. Despite intensive efforts to identify drug targets and develop neuroprotective strategies, effective therapeutics for ALS remains unavailable. The identification and characterization of novel targets and pathways remain crucial in the development of ALS therapeutics. Adenosine is a major neuromodulator that actively regulates synaptic transmission. Interestingly, adenosine levels are significantly elevated in the cerebrospinal fluid (CSF) of progressing human ALS patients. In the current study, we showed that adenosine 2a receptor (A2aR), but not adenosine 1 receptor (A1R), is highly enriched in spinal (motor) neurons. A2aR expression is also selectively increased at the symptomatic onset in the spinal cords of SOD1G93A mice and end-stage human ALS spinal cords. Interestingly, we found that direct adenosine treatment is sufficient to induce embryonic stem cell-derived motor neuron (ESMN) cell death in cultures. Subsequent pharmacological inhibition and partial genetic ablation of A2aR (A2aR(+/-)) significantly protect ESMN from SOD1G93A(+) astrocyte-induced cell death and delay disease progression of SOD1G93A mice. Taken together, our results provide compelling novel evidence that A2aR-mediated adenosine signaling contributes to the selective spinal motor neuron degeneration observed in the SOD1G93A mouse model of ALS.

  8. Suppression of adenosine 2a receptor (A2aR)-mediated adenosine signaling improves disease phenotypes in a mouse model of amyotrophic lateral sclerosis

    PubMed Central

    Ng, Seng kah; Higashimori, Haruki; Tolman, Michaela; Yang, Yongjie

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease in which the majority of upper and lower motor neurons are degenerated. Despite intensive efforts to identify drug targets and develop neuroprotective strategies, effective therapeutics for ALS remains unavailable. The identification and characterization of novel targets and pathways remain crucial in the development of ALS therapeutics. Adenosine is a major neuromodulator that actively regulates synaptic transmission. Interestingly, adenosine levels are significantly elevated in the cerebrospinal fluid (CSF) of progressing human ALS patients. In the current study, we showed that adenosine 2a receptor (A2aR), but not adenosine 1 receptor (A1R), is highly enriched in spinal (motor) neurons. A2aR expression is also selectively increased at the symptomatic onset in the spinal cords of SOD1G93A mice and end-stage human ALS spinal cords. Interestingly, we found that direct adenosine treatment is sufficient to induce embryonic stem cell-derived motor neuron (ESMN) cell death in cultures. Subsequent pharmacological inhibition and partial genetic ablation of A2aR (A2aR+/−) significantly protect ESMN from SOD1G93A+ astrocyte-induced cell death and delay disease progression of SOD1G93A mice. Taken together, our results provide compelling novel evidence that A2aR-mediated adenosine signaling contributes to the selective spinal motor neuron degeneration observed in the SOD1G93A mouse model of ALS. PMID:25779930

  9. Membrane omega-3 fatty acids modulate the oligomerisation kinetics of adenosine A2A and dopamine D2 receptors

    PubMed Central

    Guixà-González, Ramon; Javanainen, Matti; Gómez-Soler, Maricel; Cordobilla, Begoña; Domingo, Joan Carles; Sanz, Ferran; Pastor, Manuel; Ciruela, Francisco; Martinez-Seara, Hector; Selent, Jana

    2016-01-01

    Membrane levels of docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid (ω-3 PUFA), are decreased in common neuropsychiatric disorders. DHA modulates key cell membrane properties like fluidity, thereby affecting the behaviour of transmembrane proteins like G protein-coupled receptors (GPCRs). These receptors, which have special relevance for major neuropsychiatric disorders have recently been shown to form dimers or higher order oligomers, and evidence suggests that DHA levels affect GPCR function by modulating oligomerisation. In this study, we assessed the effect of membrane DHA content on the formation of a class of protein complexes with particular relevance for brain disease: adenosine A2A and dopamine D2 receptor oligomers. Using extensive multiscale computer modelling, we find a marked propensity of DHA for interaction with both A2A and D2 receptors, which leads to an increased rate of receptor oligomerisation. Bioluminescence resonance energy transfer (BRET) experiments performed on living cells suggest that this DHA effect on the oligomerisation of A2A and D2 receptors is purely kinetic. This work reveals for the first time that membrane ω-3 PUFAs play a key role in GPCR oligomerisation kinetics, which may have important implications for neuropsychiatric conditions like schizophrenia or Parkinson’s disease. PMID:26796668

  10. Membrane omega-3 fatty acids modulate the oligomerisation kinetics of adenosine A2A and dopamine D2 receptors

    NASA Astrophysics Data System (ADS)

    Guixà-González, Ramon; Javanainen, Matti; Gómez-Soler, Maricel; Cordobilla, Begoña; Domingo, Joan Carles; Sanz, Ferran; Pastor, Manuel; Ciruela, Francisco; Martinez-Seara, Hector; Selent, Jana

    2016-01-01

    Membrane levels of docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid (ω-3 PUFA), are decreased in common neuropsychiatric disorders. DHA modulates key cell membrane properties like fluidity, thereby affecting the behaviour of transmembrane proteins like G protein-coupled receptors (GPCRs). These receptors, which have special relevance for major neuropsychiatric disorders have recently been shown to form dimers or higher order oligomers, and evidence suggests that DHA levels affect GPCR function by modulating oligomerisation. In this study, we assessed the effect of membrane DHA content on the formation of a class of protein complexes with particular relevance for brain disease: adenosine A2A and dopamine D2 receptor oligomers. Using extensive multiscale computer modelling, we find a marked propensity of DHA for interaction with both A2A and D2 receptors, which leads to an increased rate of receptor oligomerisation. Bioluminescence resonance energy transfer (BRET) experiments performed on living cells suggest that this DHA effect on the oligomerisation of A2A and D2 receptors is purely kinetic. This work reveals for the first time that membrane ω-3 PUFAs play a key role in GPCR oligomerisation kinetics, which may have important implications for neuropsychiatric conditions like schizophrenia or Parkinson’s disease.

  11. Long-lasting alterations in 5-HT2A receptor after a binge regimen of methamphetamine in mice.

    PubMed

    Chiu, Hong-Yi; Chan, Ming-Huan; Lee, Mei-Yi; Chen, Shao-Tsu; Zhan, Zih-Yi; Chen, Hwei-Hsien

    2014-10-01

    The repeated administration of methamphetamine (MA) to animals in a single-day 'binge' dosing regimen produces damage to dopamine and serotonin terminals and psychosis-like behaviours similar to those observed in MA abusers. The present study aimed to examine the effects of MA binge exposure on 5-HT2A receptors, the subtype of serotonin receptors putatively involved in psychosis. ICR male mice were treated with MA (4 × 5 mg/kg) or saline at 2 h intervals. Recognition memory and social behaviours were sequentially evaluated by a novel location recognition test, a novel object recognition test, a social interaction and a nest-building test to confirm the persistent cognitive and behavioural impairments after this dosing regimen. Subsequently, a hallucinogenic 5-HT2A/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI)-induced head-twitch, molecular and electrophysiological responses were monitored. Finally, the levels of 5-HT2C, 5-HT1A, 5-HT2A and mGlu2 receptors in the medial prefrontal cortex were determined. MA binge exposure produced recognition memory impairment, reduced social behaviours, and increased DOI-induced head-twitch response, c-Fos and Egr-2 expression and field potentials in the medial prefrontal cortex. Furthermore, MA binge exposure increased 5-HT2A and decreased mGlu2 receptor expression in the medial frontal cortex, whereas 5-HT2C and 5-HT1A receptors were unaffected. These data reveal that the increased behavioural, molecular and electrophysiological responses to DOI might be associated with an up-regulation of 5-HT2A receptors in the medial prefrontal cortex after MA binge exposure. Identifying the biochemical alterations that parallel the behavioural changes in a mouse model of MA binge exposure may facilitate targeting therapies for treatment of MA-related psychiatric disorders.

  12. 5-Hydroxytryptamine-2A receptor gene (HTR 2 A) candidate polymorphism (T 102 C): Role for human platelet function under pharmacological challenge ex vivo.

    PubMed

    Ozdener, F; Gülbas, Z; Erol, K; Ozdemir, V

    2005-01-01

    Although the environmental and life-style factors influencing individual predisposition to acute myocardial infarction (AMI) have been well documented, little is known on the identity of genetic loci that may contribute to risk for AMI. Recently, genetic studies in patients with nonfatal AMI have suggested an association with the T 102 C polymorphism in the serotonin 5-HT(2A) receptor gene (HTR 2 A). Considering the significant role of the 5-HT(2A) receptor in serotonin-induced platelet responses and the contribution of platelet (patho)physiology to thromboembolic events, we postulated that the increased susceptibility to AMI in patients with the T 102 homozygosity may be attributable, in part, to altered serotonin-mediated platelet function. In a group of healthy volunteers recruited from the Eskisehir region in central Turkey (N=37), we investigated the functional consequences of HTR 2 A T 102 C polymorphism in relation to platelet pharmacodynamics ex vivo. The platelet shape change and aggregation response to serotonin were measured with use of the platelet aggregometry and expressed as aggregometer output (mm). Because the circulating catecholamine hormone epinephrine can augment platelet aggregation, pharmacodynamic response (aggregation and its inhibition by 5-HT(2A) receptor antagonist cyproheptadine) was measured in the presence of both serotonin and epinephrine, to mimic the clinical situation in patients. The mean platelet aggregation was higher by 38% in subjects with T 10 2 homozygosity (T/T genotype, N=13) when compared with the carriers of the 102 C-allele (T/C and the C/C genotypic groups, N=24) (39.5 mm+/-12.3 vs. 28.7 mm+/-16.8, respectively) (mean+/-SD) (p<0.05). On the other hand, neither the serotonin-induced platelet shape change nor the cyproheptadine inhibition of platelet aggregation was influenced by the HTR 2 A T 102 C genetic variation (p>0.05). These findings in healthy subjects may provide a mechanistic explanation for the previously

  13. GluN2A Subunit-Containing NMDA Receptors Are the Preferential Neuronal Targets of Homocysteine

    PubMed Central

    Sibarov, Dmitry A.; Abushik, Polina A.; Giniatullin, Rashid; Antonov, Sergei M.

    2016-01-01

    Homocysteine (HCY) is an endogenous redox active amino acid, best known as contributor to various neurodegenerative disorders. Although it is known that HCY can activate NMDA receptors (NMDARs), the mechanisms of its action on receptors composed of different NMDA receptor subunits remains almost unknown. In this study, using imaging and patch clamp technique in cultured cortical neurons and heterologous expression in HEK293T cells we tested the agonist activity of HCY on NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors) and GluN1 and GluN2B subunits (GluN1/2B receptors). We demonstrate that the time courses of Ca2+ transients and membrane currents activated by HCY and NMDA in cortical neurons are drastically different. Application of HCY to cortical neurons induced responses, which in contrast to currents induced by NMDA (both in the presence of glycine) considerably decreased to steady state of small amplitude. In contrast to NMDA, HCY-activated currents at steady state were resistant to the selective GluN2B subunit inhibitor ifenprodil. In calcium-free external solution the decrease of NMDA evoked currents was abolished, suggesting the Ca2+-dependent NMDAR desensitization. Under these conditions HCY evoked currents still declined almost to the baseline suggesting Ca2+-independent desensitization. In HEK293T cells HCY activated NMDARs of GluN1/2A and GluN1/2B subunit compositions with EC50s of 9.7 ± 1.8 and 61.8 ± 8.9 μM, respectively. Recombinant GluN1/2A receptors, however, did not desensitize by HCY, whereas GluN1/2B receptors were almost fully desensitized by HCY. Thus, HCY is a high affinity agonist of NMDARs preferring the GluN1/2A subunit composition. Our data suggest that HCY induced native NMDAR currents in neurons are mainly mediated by the “synaptic type” GluN1/2A NMDARs. This implies that in hyperhomocysteinemia, a disorder with enlarged level of HCY in plasma, HCY may persistently contribute to post-synaptic responses mediated

  14. Caffeine and adenosine A(2a) receptor antagonists prevent beta-amyloid (25-35)-induced cognitive deficits in mice.

    PubMed

    Dall'Igna, Oscar P; Fett, Paulo; Gomes, Marcio W; Souza, Diogo O; Cunha, Rodrigo A; Lara, Diogo R

    2007-01-01

    Consumption of caffeine, an adenosine receptor antagonist, was found to be inversely associated with the incidence of Alzheimer's disease. Moreover, caffeine protects cultured neurons against beta-amyloid-induced toxicity, an effect mimicked by adenosine A(2A) but not A(1) receptor antagonists. We now tested if caffeine administration would prevent beta-amyloid-induced cognitive impairment in mice and if this was mimicked by A(2A) receptor blockade. One week after icv administration of the 25-35 fragment of beta-amyloid (Abeta, 3 nmol), mice displayed impaired performance in both inhibitory avoidance and spontaneous alternation tests. Prolonged treatment with caffeine (1 mg/ml) had no effect alone but prevented the Abeta-induced cognitive impairment in both tasks when associated with acute caffeine (30 mg/kg) 30 min treatment before Abeta administration. The same protective effect was observed after subchronic (4 days) treatment with daily injections of either caffeine (30 mg/kg) or the selective adenosine A(2A) receptor antagonist SCH58261 (0.5 mg/kg). This provides the first direct in vivo evidence that caffeine and A(2A) receptor antagonists afford a protection against Abeta-induced amnesia, which prompts their interest for managing Alzheimer's disease.

  15. 5-HT2A Serotonin Receptor Density in Adult Male Rats’ Hippocampus after Morphine-based Conditioned Place Preference

    PubMed Central

    Mohammadi, Rabie; Jahanshahi, Mehrdad; Jameie, Seyed Behnamedin

    2016-01-01

    Introduction: A close interaction exists between the brain opioid and serotonin (5-HT) neurotransmitter systems. Brain neurotransmitter 5-HT plays an important role in the regulation of reward-related processing. However, a few studies have investigated the potential role of 5-HT2A receptors in this behavior. Therefore, the aim of the present study was to assess the influence of morphine and Conditioned Place Preference (CPP) on the density of 5-HT2A receptor in neurons of rat hippocampal formation. Methods: Morphine (10 mg/kg, IP) was injected in male Wistar rats for 7 consecutive days (intervention group), but control rats received just normal saline (1 mL/kg, IP). We used a hotplate test of analgesia to assess induction of tolerance to analgesic effects of morphine on days 1 and 8 of injections. Later, two groups of rats were sacrificed one day after 7 days of injections, their whole brains removed, and the striatum and PFC immediately dissected. Then, the NR1 gene expression was examined with a semi-quantitative RT-PCR method. Results: Our data showed that the maximum response was obtained with 2.5 mg/kg of morphine. The density of 5-HT2A receptor in different areas of the hippocampus increased significantly at sham-morphine and CPP groups (P<0.05). On the other hand, the CPP groups had more 5-HT2A receptors than sham-morphine groups and also the sham-morphine groups had more 5-HT2A receptors than the control groups. Conclusion: We concluded that the phenomenon of conditioned place preference induced by morphine can cause a significant increase in the number of serotonin 5-HT2A receptors in neurons of all areas of hippocampus. PMID:27563418

  16. Adenosine receptor targets for pain.

    PubMed

    Sawynok, J

    2016-12-03

    The main focus for the development of adenosine targets as analgesics to date has been A1Rs due to its antinociceptive profile in various preclinical pain models. The usefulness of systemic A1R agonists may be limited by other effects (cardiovascular, motor), but enhanced selectivity for pain might occur with partial agonists, potent and highly selective agonists, or allosteric modulators. A2AR agonists exhibit some peripheral pronociceptive effects, but also act on immune cells to suppress inflammation and on spinal glia to suppress pain signaling and may be useful for inflammatory and neuropathic pain. A2BR agonists exhibit peripheral proinflammatory effects on immune cells, but also spinal antinociceptive effects similar to A2AR agonists. A3Rs are now demonstrated to produce antinociception in several preclinical neuropathic pain models, with mechanistic actions on glial cells, and may be useful for neuropathic pain. Endogenous adenosine levels can be augmented by inhibition of metabolism (via adenosine kinase) or increased generation (via nucleotidases), and these approaches have implications for pain. Endogenous adenosine contributes to antinociception by several pharmacological agents, herbal remedies, acupuncture, transcutaneous electrical nerve stimulation, exercise, joint mobilization, and water immersion via spinal and/or peripheral effects, such that this system appears to constitute a major pain regulatory system. Finally, caffeine inhibits A1-, A2A- and A3Rs with similar potency, and dietary caffeine intake will need attention in trials of: (a) agonists and/or modulators acting at these receptors, (b) some pharmacological and herbal analgesics, and (c) manipulations that enhance endogenous adenosine levels, all of which are inhibited by caffeine and/or A1R antagonists in preclinical studies. All adenosine receptors have effects on spinal glial cells in regulating nociception, and gender differences in the involvement of such cells in chronic

  17. Regulation of nuclear TDP-43 by NR2A-containing NMDA receptors and PTEN

    PubMed Central

    Zheng, Mei; Liao, Mingxia; Cui, Tianyuan; Tian, Honglin; Fan, Dong-Sheng; Wan, Qi

    2012-01-01

    The dysfunction of TAR DNA-binding protein-43 (TDP-43) is implicated in neurodegenerative diseases. However, the function of TDP-43 is not fully elucidated. Here we show that the protein level of endogenous TDP-43 in the nucleus is increased in mouse cortical neurons in the early stages, but return to basal level in the later stages after glutamate accumulation-induced injury. The elevation of TDP-43 results from a downregulation of phosphatase and tensin homolog (PTEN). We further demonstrate that activation of NR2A-containing NMDA receptors (NR2ARs) leads to PTEN downregulation and subsequent reduction of PTEN import from the cytoplasm to the nucleus after glutamate accumulation. The decrease of PTEN in the nucleus contributes to its reduced association with TDP-43, and thereby mediates the elevation of nuclear TDP-43. We provide evidence that the elevation of nuclear TDP-43, mediated by NR2AR activation and PTEN downregulation, confers protection against cortical neuronal death in the late stages after glutamate accumulation. Thus, this study reveals a NR2AR–PTEN–TDP-43 signaling pathway by which nuclear TDP-43 promotes neuronal survival. These results suggest that upregulation of nuclear TDP-43 represents a self-protection mechanism to delay neurodegeneration in the early stages after glutamate accumulation and that prolonging the upregulation process of nuclear TDP-43 might have therapeutic significance. PMID:22526419

  18. The Triggering Receptor Expressed on Myeloid Cells 2: A Molecular Link of Neuroinflammation and Neurodegenerative Diseases*

    PubMed Central

    Walter, Jochen

    2016-01-01

    The triggering receptor expressed on myeloid cells (TREM) 2 is a member of the immunoglobulin superfamily of receptors and mediates signaling in immune cells via engagement of its co-receptor DNAX-activating protein of 12 kDa (DAP12). Homozygous mutations in TREM2 or DAP12 cause Nasu-Hakola disease, which is characterized by bone abnormalities and dementia. Recently, a variant of TREM2 has also been associated with an increased risk for Alzheimer disease. The selective expression of TREM2 on immune cells and its association with different forms of dementia indicate a contribution of this receptor in common pathways of neurodegeneration. PMID:26694609

  19. [Beta]-Adrenergic Receptor Activation Rescues Theta Frequency Stimulation-Induced LTP Deficits in Mice Expressing C-Terminally Truncated NMDA Receptor GluN2A Subunits

    ERIC Educational Resources Information Center

    Moody, Teena D.; Watabe, Ayako M.; Indersmitten, Tim; Komiyama, Noboru H.; Grant, Seth G. N.; O'Dell, Thomas J.

    2011-01-01

    Through protein interactions mediated by their cytoplasmic C termini the GluN2A and GluN2B subunits of NMDA receptors (NMDARs) have a key role in the formation of NMDAR signaling complexes at excitatory synapses. Although these signaling complexes are thought to have a crucial role in NMDAR-dependent forms of synaptic plasticity such as long-term…

  20. A differential role for the adenosine A2A receptor in opiate reinforcement vs opiate-seeking behavior.

    PubMed

    Brown, Robyn Mary; Short, Jennifer Lynn; Cowen, Michael Scott; Ledent, Catherine; Lawrence, Andrew John

    2009-03-01

    The adenosine A(2A) receptor is specifically enriched in the medium spiny neurons that make up the 'indirect' output pathway from the ventral striatum, a structure known to have a crucial, integrative role in processes such as reward, motivation, and drug-seeking behavior. In the present study we investigated the impact of adenosine A(2A) receptor deletion on behavioral responses to morphine in a number of reward-related paradigms. The acute, rewarding effects of morphine were evaluated using the conditioned place preference paradigm. Operant self-administration of morphine on both fixed and progressive ratio schedules as well as cue-induced drug-seeking was assessed. In addition, the acute locomotor response to morphine as well as sensitization to morphine was evaluated. Decreased morphine self-administration and breakpoint in A(2A) knockout mice was observed. These data support a decrease in motivation to consume the drug, perhaps reflecting diminished rewarding effects of morphine in A(2A) knockout mice. In support of this finding, a place preference to morphine was not observed in A(2A) knockout mice but was present in wild-type mice. In contrast, robust cue-induced morphine-seeking behavior was exhibited by both A(2A) knockout and wild-type mice after a period of withdrawal. The acute locomotor response to morphine in the A(2A) knockout was similar to wild-type mice, yet A(2A) knockout mice did not display tolerance to chronic morphine under the present paradigm. Both genotypes display locomotor sensitization to morphine, implying a lack of a role for the A(2A) receptor in the drug-induced plasticity necessary for the development or expression of sensitization. Collectively, these data suggest a differential role for adenosine A(2A) receptors in opiate reinforcement compared to opiate-seeking.

  1. An Anti-Parkinson’s Disease Drug via Targeting Adenosine A2A Receptor Enhances Amyloid-β Generation and γ-Secretase Activity

    PubMed Central

    Li, Xiaohang; Wang, Xin; Zhou, Yue; Yang, Wenjuan; Chen, Ming; Zhao, Jian; Pei, Gang

    2016-01-01

    γ-secretase mediates the intramembranous proteolysis of amyloid precursor protein (APP) and determines the generation of Aβ which is associated with Alzheimer’s disease (AD). Here we identified that an anti-Parkinson’s disease drug, Istradefylline, could enhance Aβ generation in various cell lines and primary neuronal cells of APP/PS1 mouse. Moreover, the increased generation of Aβ42 was detected in the cortex of APP/PS1 mouse after chronic treatment with Istradefylline. Istradefylline promoted the activity of γ-secretase which could lead to increased Aβ production. These effects of Istradefylline were reduced by the knockdown of A2AR but independent of A2AR-mediated G protein- or β-arrestin-dependent signal pathway. We further observed that A2AR colocalized with γ-secretase in endosomes and physically interacted with the catalytic subunit presenilin-1 (PS1). Interestingly, Istradefylline attenuated the interaction in time- and dosage-dependent manners. Moreover the knockdown of A2AR which in theory would release PS1 potentiated both Aβ generation and γ-secretase activity. Thus, our study implies that the association of A2AR could modulate γ-secretase activity. Istradefylline enhance Aβ generation and γ-secretase activity possibly via modulating the interaction between A2AR and γ-secretase, which may bring some undesired effects in the central nervous system (CNS). PMID:27835671

  2. 5-HT2A receptor antagonists improve motor impairments in the MPTP mouse model of Parkinson's disease.

    PubMed

    Ferguson, Marcus C; Nayyar, Tultul; Deutch, Ariel Y; Ansah, Twum A

    2010-01-01

    Clinical observations have suggested that ritanserin, a 5-HT(2A/C) receptor antagonist may reduce motor deficits in persons with Parkinson's Disease (PD). To better understand the potential antiparkinsonian actions of ritanserin, we compared the effects of ritanserin with the selective 5-HT(2A) receptor antagonist M100907 and the selective 5-HT(2C) receptor antagonist SB 206553 on motor impairments in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP-treated mice exhibited decreased performance on the beam-walking apparatus. These motor deficits were reversed by acute treatment with L-3,4-dihydroxyphenylalanine (levodopa). Both the mixed 5-HT(2A/C) antagonist ritanserin and the selective 5-HT(2A) antagonist M100907 improved motor performance on the beam-walking apparatus. In contrast, SB 206553 was ineffective in improving the motor deficits in MPTP-treated mice. These data suggest that 5-HT(2A) receptor antagonists may represent a novel approach to ameliorate motor symptoms of Parkinson's disease.

  3. The antidepressant 5-HT2A receptor antagonists pizotifen and cyproheptadine inhibit serotonin-enhanced platelet function.

    PubMed

    Lin, Olivia A; Karim, Zubair A; Vemana, Hari Priya; Espinosa, Enma V P; Khasawneh, Fadi T

    2014-01-01

    There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their

  4. MDMA Increases Excitability in the Dentate Gyrus: Role of 5HT2A Receptor Induced PGE2 Signaling

    PubMed Central

    Collins, Stuart A.; Huff, Courtney; Chiaia, Nicolas; Gudelsky, Gary A.; Yamamoto, Bryan K.

    2015-01-01

    MDMA is a widely abused psychostimulant which causes release of serotonin in various forebrain regions. Recently, we reported that MDMA increases extracellular glutamate concentrations in the dentate gyrus, via activation of 5HT2A receptors. We examined the role of prostaglandin signaling in mediating the effects of 5HT2A receptor activation on the increases in extracellular glutamate and the subsequent long-term loss of parvalbumin interneurons in the dentate gyrus caused by MDMA. Administration of MDMA into the dentate gyrus of rats increased PGE2 concentrations which was prevented by coadministration of MDL100907, a 5HT2A receptor antagonist. MDMA-induced increases in extracellular glutamate were inhibited by local administration of SC-51089, an inhibitor of the EP1 prostaglandin receptor. Systemic administration of SC-51089 during injections of MDMA prevented the decreases in parvalbumin interneurons observed 10 days later. The loss of parvalbumin immunoreactivity after MDMA exposure coincided with a decrease in paired-pulse inhibition and afterdischarge threshold in the dentate gyrus. These changes were prevented by inhibition of EP1 and 5HT2A receptors during MDMA. Additional experiments revealed an increased susceptibility to kainic acid-induced seizures in MDMA treated rats which could be prevented with SC51089 treatments during MDMA exposure. Overall, these findings suggest that 5HT2A receptors mediate MDMA-induced PGE2 signaling and subsequent increases in glutamate. This signaling mediates parvalbumin cell losses as well as physiologic changes in the dentate gyrus, suggesting that the lack of the inhibition provided by these neurons increases the excitability within the dentate gyrus of MDMA treated rats. PMID:26670377

  5. Controlling the Dissociation of Ligands from the Adenosine A2A Receptor through Modulation of Salt Bridge Strength.

    PubMed

    Segala, Elena; Guo, Dong; Cheng, Robert K Y; Bortolato, Andrea; Deflorian, Francesca; Doré, Andrew S; Errey, James C; Heitman, Laura H; IJzerman, Adriaan P; Marshall, Fiona H; Cooke, Robert M

    2016-07-14

    The association and dissociation kinetics of ligands binding to proteins vary considerably, but the mechanisms behind this variability are poorly understood, limiting their utilization for drug discovery. This is particularly so for G protein-coupled receptors (GPCRs) where high resolution structural information is only beginning to emerge. Engineering the human A2A adenosine receptor has allowed structures to be solved in complex with the reference compound ZM241385 and four related ligands at high resolution. Differences between the structures are limited, with the most pronounced being the interaction of each ligand with a salt bridge on the extracellular side of the receptor. Mutagenesis experiments confirm the role of this salt bridge in controlling the dissociation kinetics of the ligands from the receptor, while molecular dynamics simulations demonstrate the ability of ligands to modulate salt bridge stability. These results shed light on a structural determinant of ligand dissociation kinetics and identify a means by which this property may be optimized.

  6. Chronic treatment with LY341495 decreases 5-HT(2A) receptor binding and hallucinogenic effects of LSD in mice.

    PubMed

    Moreno, José L; Holloway, Terrell; Rayannavar, Vinayak; Sealfon, Stuart C; González-Maeso, Javier

    2013-03-01

    Hallucinogenic drugs, such as lysergic acid diethylamide (LSD), mescaline and psilocybin, alter perception and cognitive processes. All hallucinogenic drugs have in common a high affinity for the serotonin 5-HT(2A) receptor. Metabotropic glutamate 2/3 (mGlu2/3) receptor ligands show efficacy in modulating the cellular and behavioral responses induced by hallucinogenic drugs. Here, we explored the effect of chronic treatment with the mGlu2/3 receptor antagonist 2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)-propionic acid (LY341495) on the hallucinogenic-like effects induced by LSD (0.24mg/kg). Mice were chronically (21 days) treated with LY341495 (1.5mg/kg), or vehicle, and experiments were carried out one day after the last injection. Chronic treatment with LY341495 down-regulated [(3)H]ketanserin binding in somatosensory cortex of wild-type, but not mGlu2 knockout (KO), mice. Head-twitch behavior, and expression of c-fos, egr-1 and egr-2, which are responses induced by hallucinogenic 5-HT(2A) agonists, were found to be significantly decreased by chronic treatment with LY341495. These findings suggest that repeated blockade of the mGlu2 receptor by LY341495 results in reduced 5-HT(2A) receptor-dependent hallucinogenic effects of LSD.

  7. Adolescent anabolic-androgenic steroid exposure alters lateral anterior hypothalamic serotonin-2A receptors in aggressive male hamsters.

    PubMed

    Schwartzer, Jared J; Ricci, Lesley A; Melloni, Richard H

    2009-05-16

    Chronic anabolic-androgenic steroid (AAS) treatment during adolescence facilitates offensive aggression in male Syrian hamsters (Mesocricetus auratus). Serotonin (5-HT) modulates aggressive behavior and has been shown to be altered after chronic treatment with AAS. Furthermore, 5-HT type 2 receptors have been implicated in the control of aggression. For example, treatment with 5-HT(2A) receptor antagonists suppress the generation of the offensive aggressive phenotype. However, it is unclear whether these receptors are sensitive to adolescent AAS exposure. The current study assessed whether treatment with AAS throughout adolescence influenced the immunohistochemical localization of 5-HT(2A) in areas of the hamster brain implicated in the control of aggression. Hamsters were administered AAS (5.0 mg/kg) each day throughout adolescence, scored for offensive aggression, and then examined for differences in 5-HT(2A)-immunoreactivity (5-HT(2A)-ir). When compared with non-aggressive oil-treated controls, aggressive AAS-treated hamsters showed significant increases in 5-HT(2A)-ir fibers in the lateral portion of the anterior hypothalamus (LAH). Further analysis revealed that AAS treatment also produced a significant increase in the number of cells expressing 5-HT(2A)-ir in the LAH. Together, these results support a role for altered 5-HT(2A) expression and further implicate the LAH as a central brain region important in the control of adolescent AAS-induced offensive aggression.

  8. Involvement of 5-HT(2A/2B/2C) receptors on memory formation: simple agonism, antagonism, or inverse agonism?

    PubMed

    Meneses, Alfredo

    2002-12-01

    1. The 5-HT2 receptors subdivision into the 5-HT(2A/2B/2C) subtypes along with the advent of the selective antagonists has allowed a more detailed investigation on the role and therapeutic significance of these subtypes in cognitive functions. The present study further analyzed the 5-HT2 receptors role on memory consolidation. 2. The SB-200646 (a selective 5-HT(2B/2C) receptor antagonist) and LY215840 (a nonselective 5-HT(2/7) receptor antagonist) posttraining administration had no effect on an autoshaped memory consolidation. However, both drugs significantly and differentially antagonized the memory impairments induced by 1-(3-chlorophenyl)piperazine (mCPP), 1-naphtyl-piperazine (1-NP), mesulergine, or N-(3-trifluoromethylphenyl) piperazine (TFMPP). 3. In contrast, SB-200646 failed to modify the facilitatory procognitive effect produced by (+/-)-2.5-dimethoxy-4-iodoamphetamine (DOI) or ketanserin, which were sensitive to MDL100907 (a selective 5-HT2A receptor antagonist) and to a LY215840 high dose. 4. Finally, SB-200646 reversed the learning deficit induced by dizocilpine, but not that by scopolamine: while SB-200646 and MDL100907 coadministration reversed memory deficits induced by both drugs. 5. It is suggested that 5-HT(2B/2C) receptors might be involved on memory formation probably mediating a suppressive or constraining action. Whether the drug-induced memory impairments in this study are explained by simple agonism, antagonism, or inverse agonism at 5-HT2 receptors remains unclear at this time. 6. Notably, the 5-HT2 receptor subtypes blockade may provide some benefit to reverse poor memory consolidation conditions associated with decreasedcholinergic, glutamatergic, and/or serotonergic neurotransmission.

  9. Detection of new biased agonists for the serotonin 5-HT2A receptor: modeling and experimental validation.

    PubMed

    Martí-Solano, Maria; Iglesias, Alba; de Fabritiis, Gianni; Sanz, Ferran; Brea, José; Loza, M Isabel; Pastor, Manuel; Selent, Jana

    2015-04-01

    Detection of biased agonists for the serotonin 5-HT2A receptor can guide the discovery of safer and more efficient antipsychotic drugs. However, the rational design of such drugs has been hampered by the difficulty detecting the impact of small structural changes on signaling bias. To overcome these difficulties, we characterized the dynamics of ligand-receptor interactions of known biased and balanced agonists using molecular dynamics simulations. Our analysis revealed that interactions with residues S5.46 and N6.55 discriminate compounds with different functional selectivity. Based on our computational predictions, we selected three derivatives of the natural balanced ligand serotonin and experimentally validated their ability to act as biased agonists. Remarkably, our approach yielded compounds promoting an unprecedented level of signaling bias at the 5-HT2A receptor, which could help interrogate the importance of particular pathways in conditions like schizophrenia.

  10. Sustained Suppression of Hyperalgesia during Latent Sensitization by μ-, δ-, and κ-opioid receptors and α2A Adrenergic Receptors: Role of Constitutive Activity

    PubMed Central

    Walwyn, Wendy M.; Chen, Wenling; Kim, Hyeyoung; Minasyan, Ani; Ennes, Helena S.; McRoberts, James A.

    2016-01-01

    Many chronic pain disorders alternate between bouts of pain and periods of remission. The latent sensitization model reproduces this in rodents by showing that the apparent recovery (“remission”) from inflammatory or neuropathic pain can be reversed by opioid antagonists. Therefore, this remission represents an opioid receptor-mediated suppression of a sustained hyperalgesic state. To identify the receptors involved, we induced latent sensitization in mice and rats by injecting complete Freund's adjuvant (CFA) in the hindpaw. In WT mice, responses to mechanical stimulation returned to baseline 3 weeks after CFA. In μ-opioid receptor (MOR) knock-out (KO) mice, responses did not return to baseline but partially recovered from peak hyperalgesia. Antagonists of α2A-adrenergic and δ-opioid receptors reinstated hyperalgesia in WT mice and abolished the partial recovery from hyperalgesia in MOR KO mice. In rats, antagonists of α2A adrenergic and μ-, δ-, and κ-opioid receptors reinstated hyperalgesia during remission from CFA-induced hyperalgesia. Therefore, these four receptors suppress hyperalgesia in latent sensitization. We further demonstrated that suppression of hyperalgesia by MORs was due to their constitutive activity because of the following: (1) CFA-induced hyperalgesia was reinstated by the MOR inverse agonist naltrexone (NTX), but not by its neutral antagonist 6β-naltrexol; (2) pro-enkephalin, pro-opiomelanocortin, and pro-dynorphin KO mice showed recovery from hyperalgesia and reinstatement by NTX; (3) there was no MOR internalization during remission; (4) MORs immunoprecipitated from the spinal cord during remission had increased Ser375 phosphorylation; and (5) electrophysiology recordings from dorsal root ganglion neurons collected during remission showed constitutive MOR inhibition of calcium channels. SIGNIFICANCE STATEMENT Chronic pain causes extreme suffering to millions of people, but its mechanisms remain to be unraveled. Latent

  11. (±)-Nantenine analogs as antagonists at human 5-HT2A receptors: C1 and flexible congeners

    PubMed Central

    Chaudhary, Sandeep; Pecic, Stevan; LeGendre, Onica; Navarro, Hérnan A.; Harding, Wayne W.

    2009-01-01

    C1 and flexible analogs of (±)-nantenine were synthesized and evaluated for antagonist activity at human 5-HT2A receptors in a calcium mobilization assay. This work has resulted in the identification of the most potent 5-HT2A antagonist known based on an aporphine. Our results also suggest that the C1 position may be a key site for increasing 5-HT2A antagonist activity in this compound series. In addition, the structural rigidity of the aporphine core appears to be required for nantenine to function as a 5-HT2A antagonist. PMID:19328689

  12. Hippocampal serotonin-2A receptor-immunoreactive neurons density increases after testosterone therapy in the gonadectomized male mice

    PubMed Central

    Nikmahzar, Emsehgol; Ghaemi, Amir; Naseri, Gholam Reza; Moharreri, Ali Reza; Lotfinia, Ahmad Ali

    2016-01-01

    The change of steroid levels may also exert different modulatory effects on the number and class of serotonin receptors present in the plasma membrane. The effects of chronic treatment of testosterone for anxiety were examined and expression of 5-HT2A serotonergic receptor, neuron, astrocyte, and dark neuron density in the hippocampus of gonadectomized male mice was determined. Thirty-six adult male NMRI mice were randomly divided into six groups: intact-no testosterone treatment (No T), gonadectomy (GDX)-No T, GDX-Vehicle, GDX-6.25 mg/kg testosterone (T), GDX-12.5 mg/kg T, and GDX-25 mg/kg T. Anxiety-related behavior was evaluated using elevated plus maze apparatus. The animals were anesthetized after 48 hours after behavioral testing, and decapitated and micron slices were prepared for immunohistochemical as well as histopathological assessment. Subcutaneous injection of testosterone (25 mg/kg) may induce anxiogenic-like behavior in male mice. In addition, immunohistochemical data reveal reduced expression of 5-HT2A serotonergic receptor after gonadectomy in all areas of the hippocampus. However, treatment with testosterone could increase the mean number of dark neurons as well as immunoreactive neurons in CA1 and CA3 area, dose dependently. The density of 5-HT2A receptor-immunoreactive neurons may play a crucial role in the induction of anxiety like behavior. As reduction in such receptor expression have shown to significantly enhance anxiety behaviors. However, replacement of testosterone dose dependently enhances the number of 5-HT2A receptor-immunoreactive neurons and interestingly also reduced anxiety like behaviors. PMID:28127501

  13. High salt diet exacerbates vascular contraction in the absence of adenosine A2A receptor

    PubMed Central

    Pradhan, Isha; Zeldin, Darryl C.; Ledent, Catherine; Mustafa, S. Jamal; Falck, John R.; Nayeem, Mohammed A

    2014-01-01

    High salt (4%NaCl, HS) diet modulates adenosine-induced vascular response through adenosine A2A-receptor (A2AAR). Evidence suggests A2AAR stimulates cyp450-epoxygenases, leading to epoxyeicosatrienoic acids (EETs) generation. The aim of this study was to understand the vascular reactivity to HS and underlying signaling mechanism in the presence or absence of A2AAR. Therefore, we hypothesized that HS enhances adenosine-induced relaxation through EETs in A2AAR+/+, but exaggerates contraction in A2AAR−/−. Organ-bath and Western-blot experiments were conducted in HS and normal salt (NS, 0.18% NaCl)-fed A2AAR+/+ and A2AAR−/− mice aortae. HS produced concentration-dependent relaxation to non-selective adenosine analog, NECA in A2AAR+/+, whereas contraction was observed in A2AAR−/− mice and this was attenuated by A1AR antagonist (DPCPX). CGS-21680 (selective A2AAR-agonist) enhanced relaxation in HS-A2AAR+/+ vs. NS-A2AAR+/+, that was blocked by EETs antagonist (14,15-EEZE). Compared to NS, HS significantly upregulated expression of vasodilators A2AAR and cyp2c29, while vasoconstrictors A1AR and cyp4a in A2AAR+/+ were downregulated. In A2AAR−/− mice, however, HS significantly downregulated the expression of cyp2c29, while A1AR and cyp4a were upregulated compared to A2AAR+/+ mice. Hence, our data suggest that in A2AAR+/+, HS enhances A2AAR-induced relaxation through increased cyp-expoxygenases-derived EETs and decreased A1AR levels, whereas in A2AAR−/−, HS exaggerates contraction through decreased cyp-epoxygenases and increased A1AR levels. PMID:24390173

  14. A2A Adenosine Receptor (A2AAR) as a Therapeutic Target in Diabetic Retinopathy

    PubMed Central

    Ibrahim, Ahmed S.; El-shishtawy, Mamdouh M.; Zhang, Wenbo; Caldwell, Ruth B.; Liou, Gregory I.

    2011-01-01

    In diabetic retinopathy (DR), abnormalities in vascular and neuronal function are closely related to the local production of inflammatory mediators whose potential source is microglia. A2A adenosine receptor (A2AAR) has been shown to possess anti-inflammatory properties that have not been studied in DR. Here, we evaluate the role of A2AAR and its underlying signaling in retinal complications associated with diabetes. Initial studies in wild-type mice revealed that the treatment with the A2AAR agonist resulted in marked decreases in hyperglycemia-induced retinal cell death and tumor necrosis factor (TNF)-α release. To further assess the role of A2AAR in DR, we studied the effects of A2AAR ablation on diabetes-induced retinal abnormalities. Diabetic A2AAR−/− mice had significantly more terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells, TNF-α release, and intercellular adhesion molecule-1 expression compared with diabetic wild-type mice. To explore a potential mechanism by which A2AAR signaling regulates inflammation in DR, we performed additional studies using microglial cells treated with Amadori-glycated albumin, a risk factor in diabetic disorders. The results showed that activation of A2AAR attenuated Amadori-glycated albumin-induced TNF-α release in a cAMP/exchange protein directly activated by cAMP-dependent mechanism and significantly repressed the inflammatory cascade, C-Raf/extracellular signal-regulated kinase (ERK), in activated microglia. Collectively, this work provides pharmacological and genetic evidence for A2AAR signaling as a control point of cell death in DR and suggests that the retinal protective effect of A2AAR is mediated by abrogating the inflammatory response that occurs in microglia via interaction with C-Raf/ERK pathway. PMID:21514428

  15. Rapid Computational Prediction of Thermostabilizing Mutations for G Protein-Coupled Receptors

    PubMed Central

    2015-01-01

    G protein-coupled receptors (GPCRs) are highly dynamic and often denature when extracted in detergents. Deriving thermostable mutants has been a successful strategy to stabilize GPCRs in detergents, but this process is experimentally tedious. We have developed a computational method to predict the position of the thermostabilizing mutations for a given GPCR sequence. We have validated the method against experimentally measured thermostability data for single mutants of the β1-adrenergic receptor (β1AR), adenosine A2A receptor (A2AR) and neurotensin receptor 1 (NTSR1). To make these predictions we started from homology models of these receptors of varying accuracies and generated an ensemble of conformations by sampling the rigid body degrees of freedom of transmembrane helices. Then, an all-atom force field function was used to calculate the enthalpy gain, known as the “stability score” upon mutation of every residue, in these receptor structures, to alanine. For all three receptors, β1AR, A2AR, and NTSR1, we observed that mutations of hydrophobic residues in the transmembrane domain to alanine that have high stability scores correlate with high experimental thermostability. The prediction using the stability score improves when using an ensemble of receptor conformations compared to a single structure, showing that receptor flexibility is important. We also find that our previously developed LITiCon method for generating conformation ensembles is similar in performance to predictions using ensembles obtained from microseconds of molecular dynamics simulations (which is computationally hundred times slower than LITiCon). We improved the thermostability prediction by including other properties such as residue-based stress and the extent of allosteric communication by each residue in the stability score. Our method is the first step toward a computational method for rapid prediction of thermostable mutants of GPCRs. PMID:25400524

  16. Serotonin 5-HT2A receptor gene variants influence antidepressant response to repeated total sleep deprivation in bipolar depression.

    PubMed

    Benedetti, Francesco; Barbini, Barbara; Bernasconi, Alessandro; Fulgosi, Mara Cigala; Colombo, Cristina; Dallaspezia, Sara; Gavinelli, Chiara; Marino, Elena; Pirovano, Adele; Radaelli, Daniele; Smeraldi, Enrico

    2008-12-12

    5-HT2A receptor density in prefrontal cortex was associated with depression and suicide. 5-HT2A receptor gene polymorphism rs6313 was associated with 5-HT2A receptor binding potential, with the ability of individuals to use environmental support in order to prevent depression, and with sleep improvement after antidepressant treatment with mirtazapine. Studies on response to antidepressant drugs gave inconsistent results. Here we studied the effect of rs6313 on response to repeated total sleep deprivation (TSD) in 80 bipolar depressed inpatients treated with three consecutive TSD cycles (each one made of 36 h awake followed by a night of undisturbed sleep). All genotype groups showed comparable acute effects of the first TSD, but patients homozygotes for the T variant had better perceived and observed benefits from treatment than carriers of the C allele. These effects became significant after the first recovery night and during the following days, leading to a 36% higher final response rate (Hamilton depression rating<8). The higher density of postsynaptic excitatory 5-HT2A receptors in T/T homozygotes could have led to higher behavioural effects of increased 5-HT neurotransmission due to repeated TSD. Other possible mechanisms involve allostatic/homeostatic adaptation to sleep loss, and a different effect of the allele variants on epigenetic influences. Results confirm the interest for individual gene variants of the serotonin pathway in shaping clinical characteristics of depression and antidepressant response.

  17. Blunted dynamics of adenosine A2A receptors is associated with increased susceptibility to Candida albicans infection in the elderly

    PubMed Central

    Rodrigues, Lisa; Miranda, Isabel M.; Andrade, Geanne M.; Mota, Marta; Cortes, Luísa; Rodrigues, Acácio G.; Cunha, Rodrigo A.; Gonçalves, Teresa

    2016-01-01

    Opportunistic gut infections and chronic inflammation, in particular due to overgrowth of Candida albicans present in the gut microbiota, are increasingly reported in the elder population. In aged, adult and young mice, we now compared the relative intestinal over-colonization by ingested C. albicans and their translocation to other organs, focusing on the role of adenosine A2A receptors that are a main stop signal of inflammation. We report that elderly mice are more prone to over-colonization by C. albicans than adult and young mice. This fungal over-growth seems to be related with higher growth rate in intestinal lumen, independent of gut tissues invasion, but resulting in higher GI tract inflammation. We observed a particularly high colonization of the stomach, with increased rate of yeast-to-hypha transition in aged mice. We found a correlation between A2A receptor density and tissue damage due to yeast infection: comparing with young and adults, aged mice have a lower gut A2A receptor density and C. albicans infection failed to increase it. In conclusion, this study shows that aged mice have a lower ability to cope with inflammation due to C. albicans over-colonization, associated with an inability to adaptively adjust adenosine A2A receptors density. PMID:27590517

  18. Dorsal prefrontal cortical serotonin 2A receptor binding indices are differentially related to individual scores on harm avoidance.

    PubMed

    Baeken, Chris; Bossuyt, Axel; De Raedt, Rudi

    2014-02-28

    Although the serotonergic system has been implicated in healthy as well as in pathological emotional states, knowledge about its involvement in personality is limited. Earlier research on this topic suggests that post-synaptic 5-HT2A receptors could be involved in particular in frontal cortical areas. In drug-naïve healthy individuals, we examined the relationship between these 5-HT2A receptors and the temperament dimension harm avoidance (HA) using 123I-5-I-R91150 single photon emission computed tomography (SPECT). HA is a personality feature closely related to stress, anxiety and depression proneness, and it is thought to be mediated by the serotonergic system. We focused on the prefrontal cortices as these regions are frequently implicated in cognitive processes related to a variety of affective disorders. We found a positive relationship between dorsal prefrontal cortical (DPFC) 5-HT2A receptor binding indices (BI) and individual HA scores. Further, our results suggest that those individuals with a tendency to worry or to ruminate are particularly prone to display significantly higher 5-HT2A receptor BI in the left DPFC. Although we only examined psychologically healthy individuals, this relationship suggests a possible vulnerability for affective disorders.

  19. mRNA Expression and DNA Methylation Analysis of Serotonin Receptor 2A (HTR2A) in the Human Schizophrenic Brain

    PubMed Central

    Cheah, Sern-Yih; Lawford, Bruce R.; Young, Ross McD.; Morris, Charles P.; Voisey, Joanne

    2017-01-01

    Serotonin receptor 2A (HTR2A) is an important signalling factor implicated in cognitive functions and known to be associated with schizophrenia. The biological significance of HTR2A in schizophrenia remains unclear as molecular analyses including genetic association, mRNA expression and methylation studies have reported inconsistent results. In this study, we examine HTR2A expression and methylation and the interaction with HTR2A polymorphisms to identify their biological significance in schizophrenia. Subjects included 25 schizophrenia and 25 control post-mortem brain samples. Genotype and mRNA data was generated by transcriptome sequencing. DNA methylation profiles were generated for CpG sites within promoter-exon I region. Expression, genotype and methylation data were examined for association with schizophrenia. HTR2A mRNA levels were reduced by 14% (p = 0.006) in schizophrenia compared to controls. Three CpG sites were hypermethylated in schizophrenia (cg5 p = 0.028, cg7 p = 0.021, cg10 p = 0.017) and HTR2A polymorphisms rs6314 (p = 0.008) and rs6313 (p = 0.026) showed genetic association with schizophrenia. Differential DNA methylation was associated with rs6314 and rs6313. There was a strong correlation between HTR2A DNA methylation and mRNA expression. The results were nominally significant but did not survive the rigorous Benjamini-Hochberg correction for multiple testing. Differential HTR2A expression in schizophrenia in our study may be the result of the combined effect of multiple differentially methylated CpG sites. Epigenetic HTR2A regulation may alter brain function, which contributes to the development of schizophrenia. PMID:28054990

  20. mRNA Expression and DNA Methylation Analysis of Serotonin Receptor 2A (HTR2A) in the Human Schizophrenic Brain.

    PubMed

    Cheah, Sern-Yih; Lawford, Bruce R; Young, Ross McD; Morris, Charles P; Voisey, Joanne

    2017-01-04

    Serotonin receptor 2A (HTR2A) is an important signalling factor implicated in cognitive functions and known to be associated with schizophrenia. The biological significance of HTR2A in schizophrenia remains unclear as molecular analyses including genetic association, mRNA expression and methylation studies have reported inconsistent results. In this study, we examine HTR2A expression and methylation and the interaction with HTR2A polymorphisms to identify their biological significance in schizophrenia. Subjects included 25 schizophrenia and 25 control post-mortem brain samples. Genotype and mRNA data was generated by transcriptome sequencing. DNA methylation profiles were generated for CpG sites within promoter-exon I region. Expression, genotype and methylation data were examined for association with schizophrenia. HTR2A mRNA levels were reduced by 14% (p = 0.006) in schizophrenia compared to controls. Three CpG sites were hypermethylated in schizophrenia (cg5 p = 0.028, cg7 p = 0.021, cg10 p = 0.017) and HTR2A polymorphisms rs6314 (p = 0.008) and rs6313 (p = 0.026) showed genetic association with schizophrenia. Differential DNA methylation was associated with rs6314 and rs6313. There was a strong correlation between HTR2A DNA methylation and mRNA expression. The results were nominally significant but did not survive the rigorous Benjamini-Hochberg correction for multiple testing. Differential HTR2A expression in schizophrenia in our study may be the result of the combined effect of multiple differentially methylated CpG sites. Epigenetic HTR2A regulation may alter brain function, which contributes to the development of schizophrenia.

  1. A new ethyladenine antagonist of adenosine A(2A) receptors: behavioral and biochemical characterization as an antiparkinsonian drug.

    PubMed

    Pinna, Annalisa; Tronci, Elisabetta; Schintu, Nicoletta; Simola, Nicola; Volpini, Rosaria; Pontis, Silvia; Cristalli, Gloria; Morelli, Micaela

    2010-03-01

    Adenosine A(2A) receptor antagonists have emerged as an attractive non-dopaminergic target in clinical trials aimed at evaluating improvement in motor deficits in Parkinson's disease (PD). Moreover, preclinical studies suggest that A(2A) receptor antagonists may slow the course of the underlying neurodegeneration of dopaminergic neurons. In this study, we evaluated the efficacy of the new adenosine A(2A) receptor antagonist 8-ethoxy-9-ethyladenine (ANR 94) in parkinsonian models of akinesia and tremor. In addition, induction of the immediate early gene zif-268, and neuroprotective and anti-inflammatory effects of ANR 94 were evaluated. ANR 94 was effective in reversing parkinsonian tremor induced by the administration of tacrine. ANR 94 also counteracted akinesia (stepping test) and sensorimotor deficits (vibrissae-elicited forelimb-placing test), as well as potentiating l-dopa-induced contralateral turning behavior in 6-hydroxydopamine (6-OHDA) lesion model of PD. Potentiation of motor behavior in 6-OHDA-lesioned rats was not associated with increased induction of the immediate early gene zif-268 in the striatum, suggesting that ANR 94 does not induce long-term plastic changes in this structure. Finally, in a subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, ANR 94 protected nigrostriatal dopaminergic neurons from degeneration and counteracted neuroinflammatory processes by contrasting astroglial (glial fibrillary acidic protein, GFAP) and microglial (CD11b) activation. A(2A) receptor antagonism represents a uniquely realistic opportunity for improving PD treatment, since A(2A) receptor antagonists offer substantial symptomatic benefits and possibly disease-modifying activity. The characterization of ANR 94 may represent a further therapeutic opportunity for the treatment of PD with this new class of drugs.

  2. Activation of A1, A2A, or A3 adenosine receptors attenuates lung ischemia-reperfusion injury

    PubMed Central

    Gazoni, Leo M.; Walters, Dustin M.; Unger, Eric B.; Linden, Joel; Kron, Irving L.; Laubach, Victor E.

    2010-01-01

    Objective Adenosine and the activation of specific adenosine receptors are implicated in the attenuation of inflammation and organ ischemia-reperfusion (IR) injury. We hypothesized that activation of A1, A2A, or A3 adenosine receptors would provide protection against lung IR injury. Methods Using an isolated, ventilated, blood-perfused rabbit lung model, lungs underwent 18 hours cold ischemia followed by 2 hours reperfusion. Lungs were administered either vehicle, adenosine, or selective A1, A2A, or A3 receptor agonists (CCPA, ATL-313, or IB-MECA, respectively) alone or with their respective antagonists (DPCPX, ZM241385, or MRS1191) during reperfusion. Results Compared to the vehicle-treated control group, treatment with A1, A2A, or A3 agonists significantly improved function (increased lung compliance and oxygenation and decreased pulmonary artery pressure), decreased neutrophil infiltration by myeloperoxidase activity, decreased edema, and reduced TNF-α production. Adenosine treatment was also protective but not to the level of the agonists. When each agonist was paired with its respective antagonist, all protective effects were blocked. The A2A agonist reduced pulmonary artery pressure and myeloperoxidase activity and increased oxygenation to a greater degree than the A1 or A3 agonists. Conclusions Selective activation of A1, A2A, or A3 adenosine receptors provides significant protection against lung IR injury. The decreased elaboration of the potent proinflammatory cytokine, TNF-α, and decreased neutrophil sequestration likely contribute to the overall improvement in pulmonary function. These results provide evidence for the therapeutic potential of specific adenosine receptor agonists in lung transplant recipients. PMID:20398911

  3. A2A Adenosine Receptor Antagonism Reverts the Blood-Brain Barrier Dysfunction Induced by Sleep Restriction

    PubMed Central

    Hurtado-Alvarado, Gabriela; Domínguez-Salazar, Emilio; Velázquez-Moctezuma, Javier

    2016-01-01

    Chronic sleep restriction induces blood-brain barrier disruption and increases pro-inflammatory mediators in rodents. Those inflammatory mediators may modulate the blood-brain barrier and constitute a link between sleep loss and blood-brain barrier physiology. We propose that adenosine action on its A2A receptor may be modulating the blood-brain barrier dynamics in sleep-restricted rats. We administrated a selective A2A adenosine receptor antagonist (SCH58261) in sleep-restricted rats at the 10th day of sleep restriction and evaluated the blood-brain barrier permeability to dextrans coupled to fluorescein (FITC-dextrans) and Evans blue. In addition, we evaluated by western blot the expression of tight junction proteins (claudin-5, occludin, ZO-1), adherens junction protein (E-cadherin), A2A adenosine receptor, adenosine-synthesizing enzyme (CD73), and neuroinflammatory markers (Iba-1 and GFAP) in the cerebral cortex, hippocampus, basal nuclei and cerebellar vermis. Sleep restriction increased blood-brain barrier permeability to FITC-dextrans and Evans blue, and the effect was reverted by the administration of SCH58261 in almost all brain regions, excluding the cerebellum. Sleep restriction increased the expression of A2A adenosine receptor only in the hippocampus and basal nuclei without changing the expression of CD73 in all brain regions. Sleep restriction reduced the expression of tight junction proteins in all brain regions, except in the cerebellum; and SCH58261 restored the levels of tight junction proteins in the cortex, hippocampus and basal nuclei. Finally, sleep restriction induced GFAP and Iba-1 overexpression that was attenuated with the administration of SCH58261. These data suggest that the action of adenosine on its A2A receptor may have a crucial role in blood-brain barrier dysfunction during sleep loss probably by direct modulation of brain endothelial cell permeability or through a mechanism that involves gliosis with subsequent inflammation and

  4. Increased non-rapid eye movement sleep by cocaine withdrawal: possible involvement of A2A receptors.

    PubMed

    Yang, Shu-Long; Han, Jin-Yi; Kim, Yun-Bae; Nam, Sang-Yoon; Song, Sukgil; Hong, Jin Tae; Oh, Ki-Wan

    2011-02-01

    This study attempted to clarify whether cocaine withdrawal altered sleep architecture and the role of adenosine receptors in this process. Cocaine (20 mg/kg) was administered subcutaneously once per day for 7 days to rat implanted with sleep/wake recording electrode. Polygraphic signs of undisturbed sleep/wake activities were recorded for 24 h before cocaine administration (basal recording as control); withdrawal-day 1 (after 1 day of repeated cocaine administration), withdrawal-day 8 (after 8 days of repeated cocaine administration), and withdrawal-day 14 (after 14 days of repeated cocaine administration), respectively. On cocaine withdrawal-day 1, wakefulness was significantly increased, total sleep was decreased, non-rapid eye movement sleep was markedly reduced, and rapid eye movement sleep was enhanced. Sleep/wake cycles were also increased on cocaine withdrawal day 1. However, non-rapid eye movement sleep was increased on withdrawal-day 8 and 14, whereas rapid eye movement sleep was decreased and no significant changes were observed in the total sleep and sleep/wake cycles during these periods. Adenosine A(2A) receptors expression was increased on withdrawal-day 8 and 14, whereas A(1) receptors levels were reduced after 14 days of withdrawal and the A(2B) receptors remained unchanged. Our findings suggest that alterations of sleep and sleep architecture during cocaine subacute and subchronic withdrawals after repeated cocaine administration may be partially involved in A(2A) receptors over-expression in the rat hypothalamus.

  5. Deletion of striatal adenosine A(2A) receptor spares latent inhibition and prepulse inhibition but impairs active avoidance learning.

    PubMed

    Singer, Philipp; Wei, Catherine J; Chen, Jiang-Fan; Boison, Detlev; Yee, Benjamin K

    2013-04-01

    Following early clinical leads, the adenosine A(2A)R receptor (A(2A)R) has continued to attract attention as a potential novel target for treating schizophrenia, especially against the negative and cognitive symptoms of the disease because of A(2A)R's unique modulatory action over glutamatergic in addition to dopaminergic signaling. Through (i) the antagonistic interaction with the dopamine D(2) receptor, and (ii) the regulation of glutamate release and N-methyl-d-aspartate receptor function, striatal A(2A)R is ideally positioned to fine-tune the dopamine-glutamate balance, the disturbance of which is implicated in the pathophysiology of schizophrenia. However, the precise function of striatal A(2A)Rs in the regulation of schizophrenia-relevant behavior is poorly understood. Here, we tested the impact of conditional striatum-specific A(2A)R knockout (st-A(2A)R-KO) on latent inhibition (LI) and prepulse inhibition (PPI) - behavior that is tightly regulated by striatal dopamine and glutamate. These are two common cross-species translational tests for the assessment of selective attention and sensorimotor gating deficits reported in schizophrenia patients; and enhanced performance in these tests is associated with antipsychotic drug action. We found that neither LI nor PPI was significantly affected in st-A(2A)R-KO mice, although a deficit in active avoidance learning was identified in these animals. The latter phenotype, however, was not replicated in another form of aversive conditioning - namely, conditioned taste aversion. Hence, the present study shows that neither learned inattention (as measured by LI) nor sensory gating (as indexed by PPI) requires the integrity of striatal A(2A)Rs - a finding that may undermine the hypothesized importance of A(2A)R in the genesis and/or treatment of schizophrenia.

  6. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL.

    PubMed Central

    Walczak, H; Degli-Esposti, M A; Johnson, R S; Smolak, P J; Waugh, J Y; Boiani, N; Timour, M S; Gerhart, M J; Schooley, K A; Smith, C A; Goodwin, R G; Rauch, C T

    1997-01-01

    TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL-R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL-R2, by ligand-based affinity purification and subsequent molecular cloning. TRAIL-R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL-R2 contains two extracellular cysteine-rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell-surface-expressed TRAIL-R2, and TRAIL-induced apoptosis is inhibited by a TRAIL-R2-Fc fusion protein. TRAIL-R2 mRNA is widely expressed and the gene encoding TRAIL-R2 is located on human chromosome 8p22-21. Like TRAIL-R1, TRAIL-R2 engages a caspase-dependent apoptotic pathway but, in contrast to TRAIL-R1, TRAIL-R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family. PMID:9311998

  7. LSD and DOB: interaction with 5-HT2A receptors to inhibit NMDA receptor-mediated transmission in the rat prefrontal cortex.

    PubMed

    Arvanov, V L; Liang, X; Russo, A; Wang, R Y

    1999-09-01

    Both the phenethylamine hallucinogen (-)-1-2, 5-dimethoxy-4-bromophenyl-2-aminopropane (DOB), a selective serotonin 5-HT2A,2C receptor agonist, and the indoleamine hallucinogen D-lysergic acid diethylamide (LSD, which binds to 5-HT1A, 1B, 1D, 1E, 1F, 2A, 2C, 5, 6, 7, dopamine D1 and D2, and alpha1 and alpha2 adrenergic receptors), but not their non-hallucinogenic congeners, inhibited N-methyl-D-aspartate (NMDA)-induced inward current and NMDA receptor-mediated synaptic responses evoked by electrical stimulation of the forceps minor in pyramidal cells of the prefrontal cortical slices. The inhibitory effect of hallucinogens was mimicked by 5-HT in the presence of selective 5-HT1A and 5-HT3 receptor antagonists. The inhibitory action of DOB, LSD and 5-HT on the NMDA transmission was blocked by the 5-HT2A receptor antagonists R-(+)-alpha-(2, 3-dimethoxyphenil)-1-[4-fluorophenylethyl]-4-piperidineme thanol (M100907) and ketanserin. However, at low concentrations, when both LSD and DOB by themselves only partially depressed the NMDA response, they blocked the inhibitory effect of 5-HT, suggesting a partial agonist action. Whereas N-(4-aminobutyl)-5-chloro-2-naphthalenesulphonamide (W-7, a calmodulin antagonist) and N-[2-[[[3-(4'-chlorophenyl)- 2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4'-methoxy-b enzenesulphonamide phosphate (KN-93, a Ca2+/CaM-KII inhibitor), but not the negative control 2-[N-4'methoxybenzenesulphonyl]amino-N-(4'-chlorophenyl)-2-propeny l-N -methylbenzylamine phosphate (KN-92), blocked the inhibitory action of LSD and DOB, the selective protein kinase C inhibitor chelerythrine was without any effect. We conclude that phenethylamine and indoleamine hallucinogens may exert their hallucinogenic effect by interacting with 5-HT2A receptors via a Ca2+/CaM-KII-dependent signal transduction pathway as partial agonists and modulating the NMDA receptors-mediated sensory, perceptual, affective and cognitive processes.

  8. Striatal adenosine A2A and cannabinoid CB1 receptors form functional heteromeric complexes that mediate the motor effects of cannabinoids.

    PubMed

    Carriba, Paulina; Ortiz, Oskar; Patkar, Kshitij; Justinova, Zuzana; Stroik, Jessica; Themann, Andrea; Müller, Christa; Woods, Anima S; Hope, Bruce T; Ciruela, Francisco; Casadó, Vicent; Canela, Enric I; Lluis, Carme; Goldberg, Steven R; Moratalla, Rosario; Franco, Rafael; Ferré, Sergi

    2007-11-01

    The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB1 receptors, is still a matter of debate. In the present study, we report that CB1 and adenosine A2A receptors form heteromeric complexes in co-transfected HEK-293T cells and rat striatum, where they colocalize in fibrilar structures. In a human neuroblastoma cell line, CB1 receptor signaling was found to be completely dependent on A2A receptor activation. Accordingly, blockade of A2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A2A and CB1 receptors.

  9. Responding for a conditioned reinforcer, and its enhancement by nicotine, is blocked by dopamine receptor antagonists and a 5-HT(2C) receptor agonist but not by a 5-HT(2A) receptor antagonist.

    PubMed

    Guy, Elizabeth Glenn; Fletcher, Paul J

    2014-10-01

    An aspect of nicotine reinforcement that may contribute to tobacco addiction is the effect of nicotine to enhance the motivational properties of reward-associated cues, or conditioned stimuli (CSs). Several studies have now shown that nicotine enhances responding for a stimulus that has been paired with a natural reinforcer. This effect of nicotine to enhance responding for a conditioned reinforcer is likely due to nicotine-induced enhancements in mesolimbic dopaminergic activity, but this has not been directly assessed. In this study, we assessed roles for dopamine (DA) D1 or D2 receptors, and two serotonin (5-HT) receptor subtypes known to modulate DA activity, the 5-HT2C or 5-HT2A subtypes, on nicotine-enhanced responding for a conditioned reinforcer. Water-restricted rats were exposed to Pavlovian conditioning sessions, where a CS was paired with water delivery. Then, in a second phase, animals were required to perform a novel, lever-pressing response for presentations of the CS as a conditioned reinforcer. Nicotine (0.4 mg/kg) enhanced responding for the conditioned reinforcer. To examine potential roles for dopamine (DA) and serotonin (5-HT) receptors in this effect, separate groups of animals were used to assess the impact of administering the D1 receptor antagonist SCH 23390, D2 receptor antagonist eticlopride, 5-HT2C receptor agonist Ro 60-0175, or 5-HT2A receptor antagonist M100907 on nicotine-enhanced responding for conditioned reinforcement. SCH 23390, eticlopride, and Ro 60-0175 all reduced responding for conditioned reinforcement, and the ability of nicotine to enhance this effect. M100907 did not alter this behavior. Together, these studies indicate that DA D1 and D2 receptors, but not 5-HT2A receptors, contribute to the effect of nicotine to enhance responding for a conditioned reinforcer. This effect can also be modulated by 5-HT2C receptor activation.

  10. mGlu5, Dopamine D2 and Adenosine A2A Receptors in L-DOPA-induced Dyskinesias

    PubMed Central

    Morin, Nicolas; Morissette, Marc; Grégoire, Laurent; Di Paolo, Thérèse

    2016-01-01

    Patients with Parkinson’s disease (PD) receiving L-3,4-dihydroxyphenylalanine (L-DOPA, the gold-standard treatment for this disease) frequently develop abnormal involuntary movements, termed L-DOPA-induced dyskinesias (LID). Glutamate overactivity is well documented in PD and LID. An approach to manage LID is to add to L-DOPA specific agents to reduce dyskinesias such as metabotropic glutamate receptor (mGlu receptor) drugs. This article reviews the contribution of mGlu type 5 (mGlu5) receptors in animal models of PD. Several mGlu5 negative allosteric modulators acutely attenuate LID in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys and 6-hydroxydopamine(6-OHDA)-lesioned rats. Chronic administration of mGlu5 negative allosteric modulators to MPTP monkeys and 6-OHDA rats also attenuates LID while maintaining the anti-parkinsonian effect of L-DOPA. Radioligand autoradiography shows an elevation of striatal mGlu5 receptors of dyskinetic L-DOPA-treated MPTP monkeys but not in those without LID. The brain molecular correlates of the long-term effect of mGlu5 negative allosteric modulators treatments with L-DOPA attenuating development of LID was shown to extend beyond mGlu5 receptors with normalization of glutamate activity in the basal ganglia of L-DOPA-induced changes of NMDA, AMPA, mGlu2/3 receptors and VGlut2 transporter. In the basal ganglia, mGlu5 receptor negative allosteric modulators also normalize the L-DOPA-induced changes of dopamine D2 receptors, their associated signaling proteins (ERK1/2 and Akt/GSK3β) and neuropeptides (preproenkephalin, preprodynorphin) as well as the adenosine A2A receptors expression. These results show in animal models of PD reduction of LID with mGlu5 negative allosteric modulation associated with normalization of glutamate, dopamine and adenosine receptors suggesting a functional link of these receptors in chronic treatment with L-DOPA. PMID:26639458

  11. mGlu5, Dopamine D2 and Adenosine A2A Receptors in L-DOPA-induced Dyskinesias.

    PubMed

    Morin, Nicolas; Morissette, Marc; Grégoire, Laurent; Di Paolo, Thérèse

    2016-01-01

    Patients with Parkinson's disease (PD) receiving L-3,4-dihydroxyphenylalanine (L-DOPA, the gold-standard treatment for this disease) frequently develop abnormal involuntary movements, termed L-DOPA-induced dyskinesias (LID). Glutamate overactivity is well documented in PD and LID. An approach to manage LID is to add to L-DOPA specific agents to reduce dyskinesias such as metabotropic glutamate receptor (mGlu receptor) drugs. This article reviews the contribution of mGlu type 5 (mGlu5) receptors in animal models of PD. Several mGlu5 negative allosteric modulators acutely attenuate LID in 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) monkeys and 6-hydroxydopamine(6-OHDA)-lesioned rats. Chronic administration of mGlu5 negative allosteric modulators to MPTP monkeys and 6-OHDA rats also attenuates LID while maintaining the antiparkinsonian effect of L-DOPA. Radioligand autoradiography shows an elevation of striatal mGlu5 receptors of dyskinetic L-DOPA-treated MPTP monkeys but not in those without LID. The brain molecular correlates of the long-term effect of mGlu5 negative allosteric modulators treatments with L-DOPA attenuating development of LID was shown to extend beyond mGlu5 receptors with normalization of glutamate activity in the basal ganglia of L-DOPA-induced changes of NMDA, AMPA, mGlu2/3 receptors and VGlut2 transporter. In the basal ganglia, mGlu5 receptor negative allosteric modulators also normalize the L-DOPA-induced changes of dopamine D2receptors, their associated signaling proteins (ERK1/2 and Akt/GSK3β) and neuropeptides (preproenkephalin, preprodynorphin) as well as the adenosine A2A receptors expression. These results show in animal models of PD reduction of LID with mGlu5 negative allosteric modulation associated with normalization of glutamate, dopamine and adenosine receptors suggesting a functional link of these receptors in chronic treatment with L-DOPA.

  12. Binding mode similarity measures for ranking of docking poses: a case study on the adenosine A2A receptor

    NASA Astrophysics Data System (ADS)

    Anighoro, Andrew; Bajorath, Jürgen

    2016-06-01

    We report an investigation designed to explore alternative approaches for ranking of docking poses in the search for antagonists of the adenosine A2A receptor, an attractive target for structure-based virtual screening. Calculation of 3D similarity of docking poses to crystallographic ligand(s) as well as similarity of receptor-ligand interaction patterns was consistently superior to conventional scoring functions for prioritizing antagonists over decoys. Moreover, the use of crystallographic antagonists and agonists, a core fragment of an antagonist, and a model of an agonist placed into the binding site of an antagonist-bound form of the receptor resulted in a significant early enrichment of antagonists in compound rankings. Taken together, these findings showed that the use of binding modes of agonists and/or antagonists, even if they were only approximate, for similarity assessment of docking poses or comparison of interaction patterns increased the odds of identifying new active compounds over conventional scoring.

  13. A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development

    PubMed Central

    Jones, Karlie R.; Choi, Uimook; Gao, Ji-Liang; Thompson, Robert D.; Rodman, Larry E.; Malech, Harry L.; Kang, Elizabeth M.

    2017-01-01

    Agonists that target the A1, A2A, A2B and A3 adenosine receptors have potential to be potent treatment options for a number of diseases, including autoimmune diseases, cardiovascular disease and cancer. Because each of these adenosine receptors plays a distinct role throughout the body, obtaining highly specific receptor agonists is essential. Of these receptors, the adenosine A2AR and A2BR share many sequence and structural similarities but highly differ in their responses to inflammatory stimuli. Our laboratory, using a combination of specially developed cell lines and calcium release analysis hardware, has created a new and faster method for determining specificity of synthetic adenosine agonist compounds for the A2A and A2B receptors in human cells. A2A receptor expression was effectively removed from K562 cells, resulting in the development of a distinct null line. Using HIV-lentivector and plasmid DNA transfection, we also developed A2A and A2B receptor over-expressing lines. As adenosine is known to cause changes in intracellular calcium levels upon addition to cell culture, calcium release can be determined in these cell lines upon compound addition, providing a functional readout of receptor activation and allowing us to isolate the most specific adenosine agonist compounds. PMID:28317879

  14. Functional polymorphisms of the glutamate receptor N-methyl D-aspartate 2A gene are associated with heroin addiction.

    PubMed

    Zhong, H J; Huo, Z H; Dang, J; Chen, J; Zhu, Y S; Liu, J H

    2014-10-27

    Heroin dependence is a debilitating psychiatric disorder with a complex inheritance mechanism. Genetic polymorphisms in functional regions of the glutamate receptor, N-methyl D-aspartate 2A (GRIN2A) gene, which encodes the 2A subunit of the N-methyl D-aspartate (NMDA) receptor, may modulate the risk of heroin addiction. We investigated the potential association between 8 single nucleotide polymorphisms (SNPs) of the GRIN2A gene (SNPs rs3219790, rs1014531, rs8044472, rs8045712, rs9933624, rs9940680, rs1420040, and rs767749) and heroin addiction using the MassARRAY system and GeneScan. A total of 405 heroin-addicted patients and 397 healthy control subjects were recruited for this study. Statistically significant differences were observed for rs3219790 in the promoter region of the GRIN2A gene. The frequency of the (GT)26 repeats in the heroin addiction group was significantly higher than that in the control group [X(2) = 5.475, P = 0.019, odds ratio (OR) = 1.367, 95% confidence interval (CI) = 1.051-1.776]. Strong linkage disequilibrium was observed in block 1 (D' > 0.9). However, significant evidence of linkage disequilibrium was not observed between the 7 SNPs in our sample population. These data suggest that GRIN2A gene polymorphisms confer susceptibility to heroin addiction and support the hypothesis that dysfunction of GRIN2A is involved in the pathophysiological process of heroin addiction.

  15. Effects of Constant Flickering Light on Refractive Status, 5-HT and 5-HT2A Receptor in Guinea Pigs

    PubMed Central

    Li, Tao; Zheng, Changyue; Ji, Shunmei; Ma, Yuanyuan; Zhang, Shuangshuang; Zhou, Xiaodong

    2016-01-01

    Purpose To investigate the effects of constant flickering light on refractive development, the role of serotonin (i.e.5-hydroxytryptamine, 5-HT)and 5-HT2A receptor in myopia induced by flickering light in guinea pigs. Methods Forty-five guinea pigs were randomly divided into three groups: control, form deprivation myopia (FDM) and flickering light induced myopia (FLM) groups(n = 15 for each group). The right eyes of the FDM group were covered with semitransparent hemispherical plastic shells serving as eye diffusers. Guinea pigs in FLM group were raised with illumination of a duty cycle of 50% at a flash frequency of 0.5Hz. The refractive status, axial length (AL), corneal radius of curvature(CRC) were measured by streak retinoscope, A-scan ultrasonography and keratometer, respectively. Ultramicroscopy images were taken by electron microscopy. The concentrations of 5-HTin the retina, vitreous body and retinal pigment epithelium (RPE) were assessed by high performance liquid chromatography, the retinal 5-HT2A receptor expression was evaluated by immunohistofluorescence and western blot. Results The refraction of FDM and FLM eyes became myopic from some time point (the 4th week and the 6th week, respectively) in the course of the experiment, which was indicated by significantly decreased refraction and longer AL when compared with the controls (p<0.05). The concentrations of 5-HT in the retina, vitreous body and RPE of FDM and FLM eyes were significantly increased in comparison with those of control eyes (both p<0.05). Similar to FDM eyes, the expression of retinal 5-HT2A receptor in FLM eyes was significantly up-regulated compared to that of control eyes (both p<0.05). Western blot analysis showed that retinal 5-HT2A receptor level elevated less in the FLM eyes than that in the FDM eyes. Moreover, the levels of norepinephrine and epinephrine in FDM and FLM groups generally decreased when compared with control groups (all p<0.05). Conclusions Constant flickering

  16. Behavioral tolerance to lysergic acid diethylamide is associated with reduced serotonin-2A receptor signaling in rat cortex.

    PubMed

    Gresch, Paul J; Smith, Randy L; Barrett, Robert J; Sanders-Bush, Elaine

    2005-09-01

    Tolerance is defined as a decrease in responsiveness to a drug after repeated administration. Tolerance to the behavioral effects of hallucinogens occurs in humans and animals. In this study, we used drug discrimination to establish a behavioral model of lysergic acid diethylamide (LSD) tolerance and examined whether tolerance to the stimulus properties of LSD is related to altered serotonin receptor signaling. Rats were trained to discriminate 60 microg/kg LSD from saline in a two-lever drug discrimination paradigm. Two groups of animals were assigned to either chronic saline treatment or chronic LSD treatment. For chronic treatment, rats from each group were injected once per day with either 130 microg/kg LSD or saline for 5 days. Rats were tested for their ability to discriminate either saline or 60 microg/kg LSD, 24 h after the last chronic injection. Rats receiving chronic LSD showed a 44% reduction in LSD lever selection, while rats receiving chronic vehicle showed no change in percent choice on the LSD lever. In another group of rats receiving the identical chronic LSD treatment, LSD-stimulated [35S]GTPgammaS binding, an index of G-protein coupling, was measured in the rat brain by autoradiography. After chronic LSD, a significant reduction in LSD-stimulated [35S]GTPgammaS binding was observed in the medial prefrontal cortex and anterior cingulate cortex. Furthermore, chronic LSD produced a significant reduction in 2,5-dimethoxy-4-iodoamphetamine-stimulated [35S]GTPgammaS binding in medial prefrontal cortex and anterior cingulate cortex, which was blocked by MDL 100907, a selective 5-HT2A receptor antagonist, but not SB206553, a 5-HT2C receptor antagonist, indicating a reduction in 5-HT2A receptor signaling. 125I-LSD binding to 5-HT2A receptors was reduced in cortical regions, demonstrating a reduction in 5-HT2A receptor density. Taken together, these results indicate that adaptive changes in LSD-stimulated serotonin receptor signaling may mediate tolerance

  17. Cartography of 5-HT1A and 5-HT2A Receptor Subtypes in Prefrontal Cortex and Its Projections.

    PubMed

    Mengod, Guadalupe; Palacios, José M; Cortés, Roser

    2015-07-15

    Since the development of chemical neuroanatomical tools in the 1960s, a tremendous wealth of information has been generated on the anatomical components of the serotonergic system, at the microscopic level in the brain including the prefrontal cortex (PFC). The PFC receives a widespread distribution of serotonin (5-hydroxytryptamine, 5-HT) terminals from the median and dorsal raphe nuclei. 5-HT receptors were first visualized using radioligand autoradiography in the late 1980s and early 1990s and showed, in contrast to 5-HT innervation, a differential distribution of binding sites associated with different 5-HT receptor subtypes. Due to the cloning of the different 5-HT receptor subtype genes in the late 1980s and early 1990s, it was possible, using in situ hybridization histochemistry, to localize cells expressing mRNA for these receptors. Double in situ hybridization histochemistry and immunohistochemistry allowed for the chemical characterization of the phenotype of cells expressing 5-HT receptors. Tract tracing technology allowed a detailed cartography of the neuronal connections of PFC and other brain areas. Based on these data, maps have been constructed that reflect our current understanding of the different circuits where 5-HT receptors can modulate the electrophysiological, pharmacological, and behavioral functions of the PFC. We will review current knowledge regarding the cellular localization of 5-HT1A and 5-HT2A receptors in mammalian PFC and their possible functions in the neuronal circuits of the PFC. We will discuss data generated in our laboratory as well as in others, focusing on localization in the pyramidal and GABAergic neuronal cell populations in different mammalian species using molecular neuroanatomy and on the connections with other brain regions.

  18. Functional expression of 5-HT{sub 2A} receptor in osteoblastic MC3T3-E1 cells

    SciTech Connect

    Hirai, Takao; Kaneshige, Kota; Kurosaki, Teruko; Nishio, Hiroaki

    2010-05-28

    In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT{sub 2} receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT{sub 2A} and 5-HT{sub 2C} receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT{sub 2A} receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT{sub 2A} receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.

  19. Discovering the mechanisms underlying serotonin (5-HT)2A and 5-HT2C receptor regulation following nicotine withdrawal in rats.

    PubMed

    Zaniewska, Magdalena; Alenina, Natalia; Wydra, Karolina; Fröhler, Sebastian; Kuśmider, Maciej; McCreary, Andrew C; Chen, Wei; Bader, Michael; Filip, Małgorzata

    2015-08-01

    We have previously demonstrated that nicotine withdrawal produces depression-like behavior and that serotonin (5-HT)2A/2C receptor ligands modulate that mood-like state. In the present study we aimed to identify the mechanisms (changes in radioligand binding, transcription or RNA-editing) related to such a behavioral outcome. Rats received vehicle or nicotine (0.4 mg/kg, s.c.) for 5 days in home cages. Brain 5-HT2A/2C receptors were analyzed on day 3 of nicotine withdrawal. Nicotine withdrawal increased [(3)H]ketanserin binding to 5-HT2A receptors in the ventral tegmental area and ventral dentate gyrus, yet decreased binding in the nucleus accumbens shell. Reduction in [(3)H]mesulergine binding to 5-HT2C receptors was seen in the ventral dentate gyrus. Profound decrease in the 5-HT2A receptor transcript level was noted in the hippocampus and ventral tegmental area. Out of five 5-HT2C receptor mRNA editing sites, deep sequencing data showed a reduction in editing at the E site and a trend toward reduction at the C site in the hippocampus. In the ventral tegmental area, a reduction for the frequency of CD 5-HT2C receptor transcript was seen. These results show that the reduction in the 5-HT2A receptor transcript level may be an auto-regulatory response to the increased receptor density in the hippocampus and ventral tegmental area during nicotine withdrawal, while decreased 5-HT2C receptor mRNA editing may explain the reduction in receptor labeling in the hippocampus. Serotonin (5-HT)2A/2C receptor ligands alleviate depression-like state in nicotine-withdrawn rats. Here, we show that the reduction in 5-HT2A receptor transcript level may be an auto-regulatory response to the increased receptor number in the hippocampus and ventral tegmental area during nicotine withdrawal, while attenuated 5-HT2C receptor mRNA editing in the hippocampus might explain reduced inverse agonist binding to 5-HT2C receptor and suggest a shift toward a population of more active receptors. 5

  20. The atypical 5-HT2 receptor mediating tachycardia in pithed rats: pharmacological correlation with the 5-HT2A receptor subtype

    PubMed Central

    Centurión, David; Ortiz, Mario I; Saxena, Pramod R; Villalón, Carlos M

    2002-01-01

    In pithed rats, 5-HT mediates tachycardia both directly (by 5-HT2 receptors) and indirectly (by a tyramine-like effect). The receptor mediating tachycardia directly has been classified as an ‘atypical' 5-HT2 receptor since it was ‘weakly' blocked by ketanserin. Moreover, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a 5-HT2 agonist, failed to mimic 5-HT-induced tachycardia. Since 5-HT2 receptors consist of 5-HT2A, 5-HT2B and 5-HT2C subtypes, this study investigated if these subtypes mediate the above response. In pithed rats, intraperitoneally (i.p.) pre-treated with reserpine (5 mg kg−1), intravenous (i.v.) administration of 5-HT, 5-methoxytryptamine (5-MeO-T), 1-(3-chlorophenyl) piperazine (mCPP) and 5-carboxamidotryptamine (5-CT) (10, 30, 100 and 300 μg kg−1 each), produced dose-dependent tachycardic responses. Interestingly, DOI (10 – 1000 μg kg−1, i.v.) induced only slight, dose-unrelated, tachycardic responses, whilst the 5-HT2C agonist, Ro 60-0175 (10 – 1000 μg kg−1, i.v.), produced a slight tachycardia only at 300 and 1000 μg kg−1. In contrast, sumatriptan and 1-(m-trifluoromethylphenyl)- piperazine (TFMPP) were inactive. The rank order of potency was: 5-HT⩾5-MeO-T> mCPP⩾5-CT⩾DOI>Ro 60-0175. The tachycardic responses to 5-HT, which remained unaffected after i.v. saline (0.3 and 1 ml kg−1) or propranolol (3 mg kg−1), were selectively blocked by the 5-HT2A antagonists ketanserin (30 and 100 μg kg−1) or spiperone (10 and 30 μg kg−1) as well as by the non-selective 5-HT2 antagonists, ritanserin (10 and 30 μg kg−1) or mesulergine (100 μg kg−1). Remarkably, these responses were unaffected by the antagonists rauwolscine (5-HT2B), SB204741 (5-HT2B/2C) or Ro 04-6790 (5-ht6) (300 and 1000 μg kg−1 each). These results suggest that the ‘atypical' 5-HT2 receptors mediating tachycardia in reserpinized pithed rats are pharmacologically similar to the 5-HT2A

  1. Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

    PubMed

    Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N

    2015-12-01

    Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

  2. Lower cortical serotonin 2A receptors in major depressive disorder, suicide and in rats after administration of imipramine.

    PubMed

    Dean, Brian; Tawadros, Nahed; Seo, Myoung Suk; Jeon, Won Je; Everall, Ian; Scarr, Elizabeth; Gibbons, Andrew

    2014-06-01

    We have attempted to replicate studies showing higher levels of serotonin 2A receptors (HTR2A) in the cortex of people with mood disorders and to determine the effects of treating rats with antidepressant drugs on levels of that receptor. In situ [3H]ketanserin binding and autoradiography was used to measure levels of HTR2A in Brodmann's area (BA) 46 and 24 from people with major depressive disorders (MDD, n = 16), bipolar disorders (BD, n = 14) and healthy controls (n = 14) as well as the central nervous system (CNS) of rats (20 per treatment arm) treated for 10 or 28 d with fluoxetine (10 mg/kg/d) or imipramine (20 mg/kg/d). Compared with controls, HTR2A were lower in BA 24, but not BA 46, from people with MDD (p = 0.005); HTR2A were not changed in BD. Levels of HTR2A were lower in BA 24 (p = 0.007), but not BA 46, from people who had died by suicide. Finally, levels of HTR2A were lower in the CNS of rats treated with imipramine, but not fluoxetine, for 28 d, but not 10 d. From our current and previous data we conclude cortical HTR2A are lower in schizophrenia, MDD, people with mood disorders who died by suicide, rats treated with some antipsychotic or some antidepressant drugs. As levels of cortical HTR2A can be affected by the aetiologies of different disorders and mechanisms of action of different drugs, a better understanding of how such changes can occur needs to be elucidated.

  3. Potential Modes of Interaction of 9-Aminomethyl-9,10-dihydroanthracene (AMDA) Derivatives with the 5-HT2A Receptor: A Ligand Structure-Affinity Relationship, Receptor Mutagenesis and Receptor Modeling Investigation⊕

    PubMed Central

    Runyon, Scott P.; Mosier, Philip D.; Roth, Bryan L.; Glennon, Richard A.; Westkaemper, Richard B.

    2011-01-01

    The effects of 3-position substitution of 9-aminomethyl-9,10-dihydroanthracene (AMDA) on 5-HT2A receptor affinity were determined and compared to a parallel series of DOB-like 1-(2,5-dimethoxyphenyl)-2-aminopropanes substituted at the 4-position. The results were interpreted within the context of 5-HT2A receptor models that suggest that members of the DOB-like series can bind to the receptor in two distinct modes that correlate with the compounds’ functional activity. Automated ligand docking and molecular dynamics suggest that all of the AMDA derivatives, the parent of which is a 5-HT2A antagonist, bind in a fashion analogous to that for the sterically demanding antagonist DOB-like compounds. The failure of the F3406.52L mutation to adversely affect the affinity of AMDA and the 3-bromo derivative is consistent with the proposed modes of orientation. Evaluation of ligand-receptor complex models suggest that a valine/threonine exchange between the 5-HT2A and D2 receptors may be the origin of selectivity for AMDA and two substituted derivatives. PMID:18847250

  4. 17β-Estradiol Induces Sulfotransferase 2A1 Expression through Estrogen Receptor α

    PubMed Central

    Li, Wei; Ning, Miaoran; Koh, Kwi Hye; Kim, Heesue

    2014-01-01

    Sulfotransferase (SULT) 2A1 catalyzes sulfonation of drugs and endogenous compounds and plays an important role in xenobiotic metabolism as well as in the maintenance of steroid and lipid homeostasis. A recent study showed that 17β-estradiol (E2) increases the mRNA levels of SULT2A1 in human hepatocytes. Here we report the underlying molecular mechanisms. E2 enhanced SULT2A1 expression in human hepatocytes and HepG2-ER cells (HepG2 stably expressing ERα). SULT2A1 induction by E2 was abrogated by antiestrogen ICI 182,780, indicating a key role of ERα in the induction. Results from deletion and mutation assays of SULT2A1 promoter revealed three cis-elements located within –257/+140 region of SULT2A1 that are potentially responsible for the induction. Chromatin immunoprecipitation assay verified the recruitment of ERα to the promoter region. Electrophoretic mobility shift assays revealed that AP-1 proteins bind to one of the cis-elements. Interestingly, SULT2A1 promoter assays using ERα mutants revealed that the DNA-binding domain of ERα is indispensable for SULT2A1 induction by E2, suggesting that direct ERα binding to the SULT2A1 promoter is also necessary for the induction. Taken together, our results indicate that E2 enhances SULT2A1 expression by both the classical and nonclassical mechanisms of ERα action. PMID:24492894

  5. Differential involvement of 5-HT(2A) receptors in the discriminative-stimulus effects of cocaine and methamphetamine.

    PubMed

    Munzar, Patrik; Justinova, Zuzana; Kutkat, Scott W; Goldberg, Steven R

    2002-02-01

    Involvement of 5-HT(2A) receptors in the discriminative-stimulus effects of cocaine versus methamphetamine was studied in Sprague Dawley rats (n=10) trained to discriminate 10 mg/kg cocaine, i.p., from saline under a fixed-ratio 10 (FR10) schedule of food presentation. The ability of (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a 5-HT(2A) receptor agonist, and ketanserin, a 5-HT(2A) receptor antagonist, to either substitute for or block the discriminative-stimulus effects of cocaine, or to shift the cocaine dose-response curve, was evaluated. DOI (0.18-1.0 mg/kg) partially substituted for the training dose of 10 mg/kg cocaine, but only at doses that decreased rates of responding. At the highest dose of DOI tested (1.0 mg/kg), there was about 65% cocaine-appropriate responding. Substitution of DOI for cocaine and DOI-induced decreases in rates of responding were completely reversed by ketanserin (3.0 mg/kg). Ketanserin (3.0 mg/kg) also produced a significant shift to the right of the cocaine dose-response curve and antagonized increases in rates of responding produced by lower doses of cocaine. Ketanserin (1.0-10.0 mg/kg), however, did not block the discriminative-stimulus effects of the training dose of cocaine. When DOI (0.3 mg/kg) was co-administered with different doses of cocaine, there was a slight leftward shift in the cocaine dose-response curve, which was not significant and appeared to reflect simple additive effects of DOI and cocaine. In contrast, the same dose of DOI (0.3 mg/kg) produced a marked and highly significant shift to the left of the methamphetamine (0.18-1.0 mg/kg) dose-response curve in the same subjects and the effects of DOI and methamphetamine were clearly more than additive. The present findings provide new evidence that there is some serotonergic modulation of cocaine's discriminative-stimulus actions, which appears to involve stimulation of 5-HT(2A) receptors. However, involvement of 5-HT(2A) receptor activity in the

  6. Adenosine A{sub 2A} receptor-dependent proliferation of pulmonary endothelial cells is mediated through calcium mobilization, PI3-kinase and ERK1/2 pathways

    SciTech Connect

    Ahmad, Aftab; Schaack, Jerome B.; White, Carl W.; Ahmad, Shama

    2013-05-10

    Highlights: •A{sub 2A} receptor-induced pulmonary endothelial growth is mediated by PI3K and ERK1/2. •Cytosolic calcium mobilization is also critical for pulmonary endothelial growth. •Effectors of A{sub 2A} receptor, like tyrosine kinases and cAMP increase PI3K/Akt signaling. •Activation of A{sub 2A} receptor can contribute to vascular remodeling. -- Abstract: Hypoxia and HIF-2α-dependent A{sub 2A} receptor expression and activation increase proliferation of human lung microvascular endothelial cells (HLMVECs). This study was undertaken to investigate the signaling mechanisms that mediate the proliferative effects of A{sub 2A} receptor. A{sub 2A} receptor-mediated proliferation of HLMVECs was inhibited by intracellular calcium chelation, and by specific inhibitors of ERK1/2 and PI3-kinase (PI3K). The adenosine A{sub 2A} receptor agonist CGS21680 caused intracellular calcium mobilization in controls and, to a greater extent, in A{sub 2A} receptor-overexpressing HLMVECs. Adenoviral-mediated A{sub 2A} receptor overexpression as well as receptor activation by CGS21680 caused increased PI3K activity and Akt phosphorylation. Cells overexpressing A{sub 2A} receptor also manifested enhanced ERK1/2 phosphorylation upon CGS21680 treatment. A{sub 2A} receptor activation also caused enhanced cAMP production. Likewise, treatment with 8Br-cAMP increased PI3K activity. Hence A{sub 2A} receptor-mediated cAMP production and PI3K and Akt phosphorylation are potential mediators of the A{sub 2A}-mediated proliferative response of HLMVECs. Cytosolic calcium mobilization and ERK1/2 phosphorylation are other critical effectors of HLMVEC proliferation and growth. These studies underscore the importance of adenosine A{sub 2A} receptor in activation of survival and proliferative pathways in pulmonary endothelial cells that are mediated through PI3K/Akt and ERK1/2 pathways.

  7. Adenosine A2A Receptor Blockade Prevents Rotenone-Induced Motor Impairment in a Rat Model of Parkinsonism

    PubMed Central

    Fathalla, Ahmed M.; Soliman, Amira M.; Ali, Mohamed H.; Moustafa, Ahmed A.

    2016-01-01

    Pharmacological studies implicate the blockade of adenosine receptorsas an effective strategy for reducing Parkinson’s disease (PD) symptoms. The objective of this study is to elucidate the possible protective effects of ZM241385 and 8-cyclopentyl-1, 3-dipropylxanthine, two selective A2A and A1 receptor antagonists, on a rotenone rat model of PD. Rats were split into four groups: vehicle control (1 ml/kg/48 h), rotenone (1.5 mg/kg/48 h, s.c.), ZM241385 (3.3 mg/kg/day, i.p) and 8-cyclopentyl-1, 3-dipropylxanthine (5 mg/kg/day, i.p). After that, animals were subjected to behavioral (stride length and grid walking) and biochemical (measuring concentration of dopamine levels using high performance liquid chromatography, HPLC). In the rotenone group, rats displayed a reduced motor activity and disturbed movement coordination in the behavioral tests and a decreased dopamine concentration as foundby HPLC. The effect of rotenone was partially prevented in the ZM241385 group, but not with 8-cyclopentyl-1,3-dipropylxanthine administration. The administration of ZM241385 improved motor function and movement coordination (partial increase of stride length and partial decrease in the number of foot slips) and an increase in dopamine concentration in the rotenone-injected rats. However, the 8-cyclopentyl-1,3-dipropylxanthine and rotenone groups were not significantly different. These results indicate that selective A2A receptor blockade by ZM241385, but not A1 receptor blockadeby 8-cyclopentyl-1,3-dipropylxanthine, may treat PD motor symptoms. This reinforces the potential use of A2A receptor antagonists as a treatment strategy for PD patients. PMID:26973484

  8. The GS Protein-coupled A2a Adenosine Receptor Controls T Cell Help in the Germinal Center.

    PubMed

    Abbott, Robert K; Silva, Murillo; Labuda, Jasmine; Thayer, Molly; Cain, Derek W; Philbrook, Phaethon; Sethumadhavan, Shalini; Hatfield, Stephen; Ohta, Akio; Sitkovsky, Michail

    2017-01-27

    T follicular helper (TFH) cells have been shown to be critically required for the germinal center (GC) reaction where B cells undergo class switch recombination and clonal selection to generate high affinity neutralizing antibodies. However, detailed knowledge of the physiological cues within the GC microenvironment that regulate T cell help is limited. The cAMP-elevating, Gs protein-coupled A2a adenosine receptor (A2aR) is an evolutionarily conserved receptor that limits and redirects cellular immunity. However, the role of A2aR in humoral immunity and B cell differentiation is unknown. We hypothesized that the hypoxic microenvironment within the GC facilitates an extracellular adenosine-rich milieu, which serves to limit TFH frequency and function, and also promotes immunosuppressive T follicular regulatory cells (TFR). In support of this hypothesis, we found that following immunization, mice lacking A2aR (A2aRKO) exhibited a significant expansion of T follicular cells, as well as increases in TFH to TFR ratio, GC T cell frequency, GC B cell frequency, and class switching of GC B cells to IgG1. Transfer of CD4 T cells from A2aRKO or wild type donors into T cell-deficient hosts revealed that these increases were largely T cell-intrinsic. Finally, injection of A2aR agonist, CGS21680, following immunization suppressed T follicular differentiation, GC B cell frequency, and class switching of GC B cells to IgG1. Taken together, these observations point to a previously unappreciated role of GS protein-coupled A2aR in regulating humoral immunity, which may be pharmacologically targeted during vaccination or pathological states in which GC-derived autoantibodies contribute to the pathology.

  9. Mutagenesis and peptide analysis of the DRY motif in the alpha2A adrenergic receptor: evidence for alternate mechanisms in G protein-coupled receptors.

    PubMed

    Chung, Duane A; Wade, Susan M; Fowler, Carol B; Woods, Danielle D; Abada, Paolo B; Mosberg, Henry I; Neubig, Richard R

    2002-05-17

    In G protein-coupled receptors (GPCRs), a conserved aspartic acid in the DRY motif at the cytoplasmic end of helix 3 regulates the transition to the active state, while the adjacent arginine is crucial for G protein activation. To examine the functions of these two residues, we made D130I and R131Q mutations in the alpha2A adrenergic receptor (AR). We demonstrate that, unlike other GPCRs, the alpha2A AR is not constitutively activated by the D130I mutation, although the mutation increases agonist affinity. While the R131Q mutation severely disrupts function, it decreases rather than increasing agonist affinity as seen in other GPCRs. We then investigated the molecular effects of the same mutations in a peptide model and showed that Arg131 is not required for peptide-mediated G protein activation. These results indicate that the alpha2A AR does not follow the conventional GPCR mechanistic paradigm with respect to the function of the DRY motif.

  10. Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

    PubMed Central

    Hothersall, J. Daniel; Guo, Dong; Sarda, Sunil; Sheppard, Robert J.; Chen, Hongming; Keur, Wesley; Waring, Michael J.; IJzerman, Adriaan P.; Hill, Stephen J.; Dale, Ian L.

    2017-01-01

    The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy. PMID:27803241

  11. Effects of serotonin-2A receptor binding and gender on personality traits and suicidal behavior in borderline personality disorder.

    PubMed

    Soloff, Paul H; Chiappetta, Laurel; Mason, Neale Scott; Becker, Carl; Price, Julie C

    2014-06-30

    Impulsivity and aggressiveness are personality traits associated with a vulnerability to suicidal behavior. Behavioral expression of these traits differs by gender and has been related to central serotonergic function. We assessed the relationships between serotonin-2A receptor function, gender, and personality traits in borderline personality disorder (BPD), a disorder characterized by impulsive-aggression and recurrent suicidal behavior. Participants, who included 33 BPD patients and 27 healthy controls (HC), were assessed for Axis I and II disorders with the Structured Clinical Interview for DSM-IV and the International Personality Disorders Examination, and with the Diagnostic Interview for Borderline Patients-Revised for BPD. Depressed mood, impulsivity, aggression, and temperament were assessed with standardized measures. Positron emission tomography with [(18)F]altanserin as ligand and arterial blood sampling was used to determine the binding potentials (BPND) of serotonin-2A receptors in 11 regions of interest. Data were analyzed using Logan graphical analysis, controlling for age and non-specific binding. Among BPD subjects, aggression, Cluster B co-morbidity, antisocial PD, and childhood abuse were each related to altanserin binding. BPND values predicted impulsivity and aggression in BPD females (but not BPD males), and in HC males (but not HC females.) Altanserin binding was greater in BPD females than males in every contrast, but it did not discriminate suicide attempters from non-attempters. Region-specific differences in serotonin-2A receptor binding related to diagnosis and gender predicted clinical expression of aggression and impulsivity. Vulnerability to suicidal behavior in BPD may be related to serotonin-2A binding through expression of personality risk factors.

  12. Adenosine A2A receptor deletion affects social behaviors and anxiety in mice: Involvement of anterior cingulate cortex and amygdala.

    PubMed

    López-Cruz, Laura; Carbó-Gas, Maria; Pardo, Marta; Bayarri, Pilar; Valverde, Olga; Ledent, Catherine; Salamone, John D; Correa, Mercè

    2017-03-15

    Blockade of adenosine A2A receptors can potentiate motivation to work for natural reinforcers such as food. Conspecific interaction is a potent natural reinforcer in social animals that can be manifested as preference for social exploration versus other sources of novel stimulation. Deficiencies in this type of motivated behavior (social withdrawal) have been seen in several pathologies such as autism and depression. However, the role of A2A receptors in motivation for social interaction has not been widely explored. Social interaction paradigms evaluate the natural preference of animals for exploring other conspecifics, and the ability to differentiate between familiar versus novel ones. Anxiety is one of the factors that can induce avoidance of social interaction. In the present study, adenosine A2A knockout (A2AKO) and wild-type (WT) mice were assessed for social and anxiety-related behaviors. c-Fos immunoreactivity was evaluated as a measure of neuronal activation in brain areas involved in different aspects of motivation and emotional processes. Although A2AKO mice showed an anxious profile, they displayed higher levels of sociability and were less sensitive to social novelty. WT mice displayed a typical pattern of social recognition 24h later, but not A2AKO mice, which explored equally both conspecifics. There were no differences between strains in aggressiveness, perseverance or social odor preferences. c-Fos immunoreactivity in A2AKO mice was higher in anterior cingulate and amygdala compared to WT mice. Thus, A2A receptors appear to be potential targets for the improvement of pathologies related to social function.

  13. Novel Alexa Fluor-488 labeled antagonist of the A(2A) adenosine receptor: Application to a fluorescence polarization-based receptor binding assay.

    PubMed

    Kecskés, Miklós; Kumar, T Santhosh; Yoo, Lena; Gao, Zhan-Guo; Jacobson, Kenneth A

    2010-08-15

    Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A(2A) adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A(2A)AR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a K(i) value of 111+/-16nM in radioligand binding using [(3)H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A(2A)AR. In a cyclic AMP functional assay, MRS5346 was shown to be an A(2A)AR antagonist. MRS5346 did not show any effect on A(1) and A(3) ARs in binding or the A(2B)AR in a cyclic AMP assay at 10microM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal following A(2A)AR binding. The FP signal was optimal with 20nM MRS5346 and 150microg protein/mL HEK293 membranes. The association and dissociation kinetic parameters were readily determined using this FP assay. The K(d) value of MRS5346 calculated from kinetic parameters was 16.5+/-4.7nM. In FP competition binding experiments using MRS5346 as a tracer, K(i) values of known AR agonists and antagonists consistently agreed with K(i) values from radioligand binding. Thus, this FP assay, which eliminates using radioisotopes, appears to be appropriate for both routine receptor binding and high-throughput screening with respect to speed of analysis, displaceable signal and precision. The approach used in the present study could be generally applicable to other GPCRs.

  14. Up-regulation of striatal adenosine A(2A) receptors with iron deficiency in rats: effects on locomotion and cortico-striatal neurotransmission.

    PubMed

    Quiroz, César; Pearson, Virginia; Gulyani, Seema; Allen, Richard; Earley, Christopher; Ferré, Sergi

    2010-07-01

    Brain iron deficiency leads to altered dopaminergic function in experimental animals, which can provide a mechanistic explanation for iron deficiency-related human sensory-motor disorders, such as Restless Legs Syndrome (RLS). However, mechanisms linking both conditions have not been determined. Considering the strong modulation exerted by adenosine on dopamine signaling, one connection could involve changes in adenosine receptor expression or function. In the striatum, presynaptic A(2A) receptors are localized in glutamatergic terminals contacting GABAergic dynorphinergic neurons and their function can be analyzed by the ability of A(2A) receptor antagonists to block the motor output induced by cortical electrical stimulation. Postsynaptic A(2A) receptors are localized in the dendritic field of GABAergic enkephalinergic neurons and their function can be analyzed by studying the ability of A(2A) receptor antagonists to produce locomotor activity and to counteract striatal ERK1/2 phosphorylation induced by cortical electrical stimulation. Increased density of striatal A(2A) receptors was found in rats fed during 3 weeks with an iron-deficient diet during the post-weaning period. In iron-deficient rats, the selective A(2A) receptor antagonist MSX-3, at doses of 1 and 3 mg/kg, was more effective at blocking motor output induced by cortical electrical stimulation (presynaptic A(2A) receptor-mediated effect) and at enhancing locomotor activation and blocking striatal ERK phosphorylation induced by cortical electrical stimulation (postsynaptic A(2A) receptor-mediated effects). These results indicate that brain iron deficiency induces a functional up-regulation of both striatal pre- and postsynaptic A(2A) receptor, which could be involved in sensory-motor disorders associated with iron deficiency such as RLS.

  15. Dipyridamole attenuates ischemia reperfusion induced acute kidney injury through adenosinergic A1 and A2A receptor agonism in rats.

    PubMed

    Puri, Nikkita; Mohey, Vinita; Singh, Manjinder; Kaur, Tajpreet; Pathak, Devendra; Buttar, Harpal Singh; Singh, Amrit Pal

    2016-04-01

    Dipyridamole (DYP) is an anti-platelet agent with marked vasodilator, anti-oxidant, and anti-inflammatory activity. The present study investigated the role of adenosine receptors in DYP-mediated protection against ischemia reperfusion-induced acute kidney injury (AKI) in rats. The rats were subjected to bilateral renal ischemia for 40 min followed by reperfusion for 24 h. The renal damage induced by ischemia reperfusion injury (IRI) was assessed by measuring creatinine clearance, blood urea nitrogen, uric acid, plasma potassium, fractional excretion of sodium, and microproteinuria in rats. The oxidative stress in renal tissues was assessed by quantification of thiobarbituric acid-reactive substances, superoxide anion generation, and reduced glutathione level. The hematoxylin-eosin staining was carried out to observe histopathological changes in renal tissues. DYP (10 and 30 mg/kg, intraperitoneal, i.p.) was administered 30 min before subjecting the rats to renal IRI. In separate groups, caffeine (50 mg/kg, i.p.), an adenosinergic A1 and A2A receptor antagonist was administered with and without DYP treatment before subjecting the rats to renal IRI. The ischemia reperfusion-induced AKI was demonstrated by significant changes in serum as well as urinary parameters, enhanced oxidative stress, and histopathological changes in renal tissues. The administration of DYP demonstrated protection against AKI. The prior treatment with caffeine abolished DYP-mediated reno-protection suggesting role of A1 and A2A adenosine receptors in DYP-mediated reno-protection in rats. It is concluded that adenosine receptors find their definite involvement in DYP-mediated anti-oxidative and reno-protective effect against ischemia reperfusion-induced AKI.

  16. MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit.

    PubMed

    Volkmann, Robert A; Fanger, Christopher M; Anderson, David R; Sirivolu, Venkata Ramana; Paschetto, Kathy; Gordon, Earl; Virginio, Caterina; Gleyzes, Melanie; Buisson, Bruno; Steidl, Esther; Mierau, Susanna B; Fagiolini, Michela; Menniti, Frank S

    2016-01-01

    GluN2A is the most abundant of the GluN2 NMDA receptor subunits in the mammalian CNS. Physiological and genetic evidence implicate GluN2A-containing receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2A-selective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)pyrazine-2-carboxamide) and MPX-007 (5-(((3-fluoro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)methylpyrazine-2-carboxamide). MPX-004 and MPX-007 inhibit GluN2A-containing NMDA receptors expressed in HEK cells with IC50s of 79 nM and 27 nM, respectively. In contrast, at concentrations that completely inhibited GluN2A activity these compounds have no inhibitory effect on GluN2B or GluN2D receptor-mediated responses in similar HEK cell-based assays. Potency and selectivity were confirmed in electrophysiology assays in Xenopus oocytes expressing GluN2A-D receptor subtypes. Maximal concentrations of MPX-004 and MPX-007 inhibited ~30% of the whole-cell current in rat pyramidal neurons in primary culture and MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices. GluN2A-selectivity at native receptors was confirmed by the finding that MPX-004 had no inhibitory effect on NMDA receptor mediated synaptic currents in cortical slices from GRIN2A knock out mice. Thus, MPX-004 and MPX-007 offer highly selective pharmacological tools to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.

  17. Cbln1 is a ligand for an orphan glutamate receptor delta2, a bidirectional synapse organizer.

    PubMed

    Matsuda, Keiko; Miura, Eriko; Miyazaki, Taisuke; Kakegawa, Wataru; Emi, Kyoichi; Narumi, Sakae; Fukazawa, Yugo; Ito-Ishida, Aya; Kondo, Tetsuro; Shigemoto, Ryuichi; Watanabe, Masahiko; Yuzaki, Michisuke

    2010-04-16

    Cbln1, secreted from cerebellar granule cells, and the orphan glutamate receptor delta2 (GluD2), expressed by Purkinje cells, are essential for synapse integrity between these neurons in adult mice. Nevertheless, no endogenous binding partners for these molecules have been identified. We found that Cbln1 binds directly to the N-terminal domain of GluD2. GluD2 expression by postsynaptic cells, combined with exogenously applied Cbln1, was necessary and sufficient to induce new synapses in vitro and in the adult cerebellum in vivo. Further, beads coated with recombinant Cbln1 directly induced presynaptic differentiation and indirectly caused clustering of postsynaptic molecules via GluD2. These results indicate that the Cbln1-GluD2 complex is a unique synapse organizer that acts bidirectionally on both pre- and postsynaptic components.

  18. The receptor EP3 to PGE2: A rational target to prevent atherothrombosis without inducing bleeding.

    PubMed

    Mawhin, Marie-Anne; Tilly, Peggy; Fabre, Jean-Etienne

    2015-09-01

    The prostanoid E2 (PGE2) is known to modulate the aggregative response of platelets to their conventional agonists such as ADP, TXA2, thrombin or collagen. Through the activation of its receptor EP3, PGE2 sensitizes platelets to their agonists but also inhibits them through its two other receptors, EP2 and EP4. In mice, the net result of these opposed actions is the EP3-mediated potentiation of platelet aggregation and the in vivo aggravation of murine atherothrombosis. Since the pathway PGE2/EP3 is not involved in murine hemostasis, we propose a "platelet EP3 paradigm" to describe this apparently paradoxical association between the facilitating impact on atherothrombosis and the unaltered hemostasis. Consistent with this paradigm, a drug blocking EP3 dramatically decreased atherothrombosis without inducing bleeding in mice. In humans, several studies did not agree on the effect of PGE2 on platelets. Reinterpreting these data with the notion of "potentiation window" and taking the platelet initial cAMP level into account reconciled these inconsistent results. Thereby, the in vitro potentiating effect of PGE2 on human platelets becomes clear. In addition, the EP3 blocking drug DG-041 abrogated the potentiating effect of PGE2 in whole human blood but did not prolong bleeding times in volunteers. Thus, the murine "platelet EP3 paradigm" would apply to humans if the aggravating role of PGE2 on atherothrombosis is shown in patients. Therefore, testing an EP3 blocker in a phase III trial would be of high interest to fulfill the unmet medical need which is to control atherothrombosis without impacting hemostasis and thus to improve the prevention of myocardial infarction.

  19. Adenosine through the A2A adenosine receptor increases IL-1β in the brain contributing to anxiety

    PubMed Central

    Chiu, Gabriel S.; Darmody, Patrick T.; Walsh, John P.; Moon, Morgan L.; Kwakwa, Kristin A.; Bray, Julie K.; McCusker, Robert H.; Freund, Gregory G.

    2014-01-01

    Anxiety is one of the most commonly reported psychiatric conditions, but its pathogenesis is poorly understood. Ailments associated with activation of the innate immune system, however, are increasingly linked to anxiety disorders. In adult male mice, we found that adenosine doubled caspase-1 activity in brain by a pathway reliant on ATP-sensitive potassium (KATP) channels, protein kinase A (PKA) and the A2A adenosine receptor (AR). In addition, adenosine-dependent activation of caspase-1 increased interleukin (IL)-1β in the brain by two-fold. Peripheral administration of adenosine in wild-type (WT) mice led to a 2.3-fold increase in caspase-1 activity in the amygdala and to a 33% and 42% reduction in spontaneous locomotor activity and food intake, respectively, that were not observed in caspase-1 knockout (KO), IL-1 receptor type 1 (IL-1R1) KO and A2A AR KO mice or in mice administered a caspase-1 inhibitor centrally. Finally, adenosine administration increased anxiety-like behaviors in WT mice by 28% in the open field test and by 55% in the elevated zero-maze. Caspase-1 KO mice, IL-1R1 KO mice, A2A AR KO mice and WT mice treated with the KATP channel blocker, glyburide, were resistant to adenosine-induced anxiety-like behaviors. Thus, our results indicate that adenosine can act as an anxiogenic by activating caspase-1 and increasing IL-1β in the brain. PMID:24907587

  20. The deubiquitinating enzyme DUB2A enhances CSF3 signalling by attenuating lysosomal routing of the CSF3 receptor.

    PubMed

    Meenhuis, Annemarie; Verwijmeren, Carola; Roovers, Onno; Touw, Ivo P

    2011-03-01

    Ubiquitination of the CSF3R [CSF3 (colony-stimulating factor 3) receptor] occurs after activated CSF3Rs are internalized and reside in early endosomes. CSF3R ubiquitination is crucial for lysosomal routing and degradation. The E3 ligase SOCS3 (suppressor of cytokine signalling 3) has been shown to play a major role in this process. Deubiquitinating enzymes remove ubiquitin moieties from target proteins by proteolytic cleavage. Two of these enzymes, AMSH [associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)] and UBPY (ubiquitin isopeptidase Y), interact with the general endosomal sorting machinery. Whether deubiquitinating enzymes control CSF3R trafficking from early towards late endosomes is unknown. In the present study, we asked whether AMSH, UBPY or a murine family of deubiquitinating enzymes could fulfil such a role. This DUB family (deubiquitin enzyme family) comprises four members (DUB1, DUB1A, DUB2 and DUB2A), which were originally described as being haematopoietic-specific and cytokine-inducible, but their function in cytokine receptor routing and signalling has remained largely unknown. We show that DUB2A expression is induced by CSF3 in myeloid 32D cells and that DUB2 decreases ubiquitination and lysosomal degradation of the CSF3R, leading to prolonged signalling. These results support a model in which CSF3R ubiquitination is dynamically controlled at the early endosome by feedback mechanisms involving CSF3-induced E3 ligase (SOCS3) and deubiquitinase (DUB2A) activities.

  1. Selecting an optimal number of binding site waters to improve virtual screening enrichments against the adenosine A2A receptor.

    PubMed

    Lenselink, Eelke B; Beuming, Thijs; Sherman, Woody; van Vlijmen, Herman W T; IJzerman, Adriaan P

    2014-06-23

    A major challenge in structure-based virtual screening (VS) involves the treatment of explicit water molecules during docking in order to improve the enrichment of active compounds over decoys. Here we have investigated this in the context of the adenosine A2A receptor, where water molecules have previously been shown to be important for achieving high enrichment rates with docking, and where the positions of some binding site waters are known from a high-resolution crystal structure. The effect of these waters (both their presence and orientations) on VS enrichment was assessed using a carefully curated set of 299 high affinity A2A antagonists and 17,337 decoys. We show that including certain crystal waters greatly improves VS enrichment and that optimization of water hydrogen positions is needed in order to achieve the best results. We also show that waters derived from a molecular dynamics simulation - without any knowledge of crystallographic waters - can improve enrichments to a similar degree as the crystallographic waters, which makes this strategy applicable to structures without experimental knowledge of water positions. Finally, we used decision trees to select an ensemble of structures with different water molecule positions and orientations that outperforms any single structure with water molecules. The approach presented here is validated against independent test sets of A2A receptor antagonists and decoys from the literature. In general, this water optimization strategy could be applied to any target with waters-mediated protein-ligand interactions.

  2. Adenosine A2A receptors and uric acid mediate protective effects of inosine against TNBS-induced colitis in rats.

    PubMed

    Rahimian, Reza; Fakhfouri, Gohar; Daneshmand, Ali; Mohammadi, Hamed; Bahremand, Arash; Rasouli, Mohammad Reza; Mousavizadeh, Kazem; Dehpour, Ahmad Reza

    2010-12-15

    Inflammatory bowel disease comprises chronic recurrent inflammation of gastrointestinal tract. This study was conducted to investigate inosine, a potent immunomodulator, in 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced chronic model of experimental colitis, and contribution of adenosine A(2A) receptors and the metabolite uric acid as possible underlying mechanisms. Experimental colitis was rendered in rats by a single colonic administration of 10 mg of TNBS. Inosine, potassium oxonate (a hepatic uricase inhibitor), SCH-442416 (a selective adenosine A(2A) receptor antagonist), inosine+potassium oxonate, or inosine+SCH-442416 were given twice daily for 7 successive days. At the end of experiment, macroscopic and histopathologic scores, colonic malondialdehyde (MDA), Tumor Necrosis Factor-alpha (TNF-α) and Interleukin-1beta (IL-1β) levels, and myeloperoxidase (MPO) activity were assessed. Plasma uric acid level was measured throughout the experiment. Both macroscopic and histological features of colonic injury were markedly ameliorated by either inosine, oxonate or inosine+oxonate. Likewise, the elevated amounts of MPO and MDA abated as well as those of TNF-α and IL-1β (P<0.05). SCH-442416 partially reversed the effect of inosine on theses markers, while inosine+oxonate showed a higher degree of protection than each treatment alone (P<.0.05). No significant difference was observed between TNBS and SCH-442416 groups. Uric acid levels were significantly higher in inosine or oxonate groups compared to control. Inosine+oxonate resulted in an even more elvelated uric acid level than each treatment alone (P<0.05). Inosine elicits notable anti-inflammatory effects on TNBS-induced colitis in rats. Uric acid and adenosine A(2A) receptors contribute to these salutary properties.

  3. Sinomenine protects against lipopolysaccharide-induced acute lung injury in mice via adenosine A(2A) receptor signaling.

    PubMed

    Li, Jun; Zhao, Li; He, Xie; Zeng, Yi-Jun; Dai, Shuang-Shuang

    2013-01-01

    Sinomenine (SIN) is a bioactive alkaloid extracted from the Chinese medicinal plant Sinomenium acutum, which is widely used in the clinical treatment of rheumatoid arthritis (RA). However, its role in acute lung injury (ALI) is unclear. In this study, we investigate the role of SIN in lipopolysaccharide (LPS)-induced ALI in mice. After ALI, lung water content and histological signs of pulmonary injury were attenuated, whereas the PaO2/FIO2 (P/F) ratios were elevated significantly in the mice pretreated with SIN. Additionally, SIN markedly inhibited inflammatory cytokine TNF-α and IL-1β expression levels as well as neutrophil infiltration in the lung tissues of the mice. Microarray analysis and real-time PCR showed that SIN treatment upregulated adenosine A(2A) receptor (A(2A)R) expression, and the protective effect of SIN was abolished in A(2A)R knockout mice. Further investigation in isolated mouse neutrophils confirmed the upregulation of A(2A)R by SIN and showed that A(2A)R-cAMP-PKA signaling was involved in the anti-inflammatory effect of SIN. Taken together, these findings demonstrate an A(2A)R-associated anti-inflammatory effect and the protective role of SIN in ALI, which suggests a potential novel approach to treat ALI.

  4. Inhibition of alpha oscillations through serotonin-2A receptor activation underlies the visual effects of ayahuasca in humans.

    PubMed

    Valle, Marta; Maqueda, Ana Elda; Rabella, Mireia; Rodríguez-Pujadas, Aina; Antonijoan, Rosa Maria; Romero, Sergio; Alonso, Joan Francesc; Mañanas, Miquel Àngel; Barker, Steven; Friedlander, Pablo; Feilding, Amanda; Riba, Jordi

    2016-07-01

    Ayahuasca is an Amazonian psychotropic plant tea typically obtained from two plants, Banisteriopsis caapi and Psychotria viridis. It contains the psychedelic 5-HT2A and sigma-1 agonist N,N-dimethyltryptamine (DMT) plus β-carboline alkaloids with monoamine-oxidase (MAO)-inhibiting properties. Although the psychoactive effects of ayahuasca have commonly been attributed solely to agonism at the 5-HT2A receptor, the molecular target of classical psychedelics, this has not been tested experimentally. Here we wished to study the contribution of the 5-HT2A receptor to the neurophysiological and psychological effects of ayahuasca in humans. We measured drug-induced changes in spontaneous brain oscillations and subjective effects in a double-blind randomized placebo-controlled study involving the oral administration of ayahuasca (0.75mg DMT/kg body weight) and the 5-HT2A antagonist ketanserin (40mg). Twelve healthy, experienced psychedelic users (5 females) participated in four experimental sessions in which they received the following drug combinations: placebo+placebo, placebo+ayahuasca, ketanserin+placebo and ketanserin+ayahuasca. Ayahuasca induced EEG power decreases in the delta, theta and alpha frequency bands. Current density in alpha-band oscillations in parietal and occipital cortex was inversely correlated with the intensity of visual imagery induced by ayahuasca. Pretreatment with ketanserin inhibited neurophysiological modifications, reduced the correlation between alpha and visual effects, and attenuated the intensity of the subjective experience. These findings suggest that despite the chemical complexity of ayahuasca, 5-HT2A activation plays a key role in the neurophysiological and visual effects of ayahuasca in humans.

  5. Emotional management and 5-HT2A receptor gene variance in patients with schizophrenia.

    PubMed

    Lo, Chi-Hsuan; Tsai, Guochuan E; Liao, Chun-Hui; Wang, Ming-Yu; Chang, Jane Pei-Chen; Tsuang, Hui-Chun; Lane, Hsien-Yuan

    2010-02-01

    Individuals with schizophrenia exhibit impaired social cognitive functions, particularly emotion management. Emotion management may be partially regulated by the serotoninergic system; the -1438 A/G polymorphism in the promoter region of the 5-HT2A gene can modulate 5-HT2A activity and is linked to certain emotional traits and anger- and aggression-related behaviors. The current study aimed to investigate whether this 5-HT2A genetic variance is associated with social cognitive function, particularly the management of emotions. One hundred and fifteen patients with chronic schizophrenia were stabilized with an optimal-dose of antipsychotic treatment. All were genotyped for the -1438 A/G polymorphism and assessed with symptom rating scales, neurocognitive instruments, and the "Managing Emotions" section of Mayer-Salovey-Caruso Emotional Intelligence Test (MSCEIT). Multiple regression showed that patients with the A/G genotype performed better than those with G/G in managing emotion (p=0.018) but did not differ from those with the A/A genotype. Regarding the two subtasks of the Managing Emotions section, the A/G heterozygotes also performed better than the G/G homozygotes in the emotion management (p=0.026) and emotional relations (p=0.027) subtasks. The results suggest that variability in the 5-HT2A gene may influence emotion management in patients with schizophrenia.

  6. Long-term estrogen therapy and 5-HT(2A) receptor binding in postmenopausal women; a single photon emission tomography (SPET) study.

    PubMed

    Compton, J; Travis, M J; Norbury, R; Erlandsson, K; van Amelsvoort, T; Daly, E; Waddington, W; Matthiasson, P; Eersels, J L H; Whitehead, M; Kerwin, R W; Ell, P J; Murphy, D G M

    2008-01-01

    Variation in estrogen level is reported by some to affect brain maturation and memory. The neurobiological basis for this may include modulation of the serotonergic system. No neuroimaging studies have directly examined the effect of extended estrogen therapy (ET), on the 5-HT(2A) receptor in human brain. We investigated the effect of long-term ET on cortical 5-HT(2A) receptor availability in postmenopausal women. In a cross-sectional study, we compared cortical 5-HT(2A) receptor availability in 17 postmenopausal ERT-naive women and 17 long-term oophorectomised estrogen-users, age- and IQ-matched using single photon emission tomography and the selective 5-HT(2A) receptor ligand (123)I-5-I-R91150. Also, we used the Revised Wechsler Memory Scale to relate memory function to 5-HT(2A) receptor availability. Never-users had significantly higher 5-HT(2A) receptor availability than estrogen-users in hippocampus (1.17 vs. 1.11, respectively, p=0.02), although this did not remain significant after correction for multiple comparisons. Hippocampal 5-HT(2A) receptor availability correlated negatively with verbal and general memory and delayed recall (r=-0.45, p=0.01; r=-0.40, p=0.02; r=-0.36, p=0.04). Right superior temporal 5-HT(2A) receptor availability correlated negatively with verbal memory (r=-0.36, p=0.04). In estrogen-users, receptor availability correlated negatively with verbal and general memory (r=-0.70, p=0.002; r=-0.69, p=0.002); and in never-users, receptor availability negatively correlated with attention and concentration (r=-0.54, p=0.02). Long-term ET may be associated with lower 5-HT(2A) receptor availability in hippocampus. This may reflect increased activity within the serotonergic pathway leading to down-regulation of post-synaptic receptor. Also, increased availability of the 5-HT(2A) receptor in hippocampus is associated with poorer memory function.

  7. Adenosine A2A receptor and ecto-5'-nucleotidase/CD73 are upregulated in hippocampal astrocytes of human patients with mesial temporal lobe epilepsy (MTLE).

    PubMed

    Barros-Barbosa, Aurora R; Ferreirinha, Fátima; Oliveira, Ângela; Mendes, Marina; Lobo, M Graça; Santos, Agostinho; Rangel, Rui; Pelletier, Julie; Sévigny, Jean; Cordeiro, J Miguel; Correia-de-Sá, Paulo

    2016-12-01

    Refractoriness to existing medications of up to 80 % of the patients with mesial temporal lobe epilepsy (MTLE) prompts for finding new antiepileptic drug targets. The adenosine A2A receptor emerges as an interesting pharmacological target since its excitatory nature partially counteracts the dominant antiepileptic role of endogenous adenosine acting via inhibitory A1 receptors. Gain of function of the excitatory A2A receptor has been implicated in a significant number of brain pathologies commonly characterized by neuronal excitotoxicity. Here, we investigated changes in the expression and cellular localization of the A2A receptor and of the adenosine-generating enzyme, ecto-5'-nucleotidase/CD73, in the hippocampus of control individuals and MTLE human patients. Western blot analysis indicates that the A2A receptor is more abundant in the hippocampus of MTLE patients compared to control individuals. Immunoreactivity against the A2A receptor predominates in astrocytes staining positively for the glial fibrillary acidic protein (GFAP). No co-localization was observed between the A2A receptor and neuronal cell markers, like synaptotagmin 1/2 (nerve terminals) and neurofilament 200 (axon fibers). Hippocampal astrogliosis observed in MTLE patients was accompanied by a proportionate increase in A2A receptor and ecto-5'-nucleotidase/CD73 immunoreactivities. Given our data, we hypothesize that selective blockade of excessive activation of astrocytic A2A receptors and/or inhibition of surplus adenosine formation by membrane-bound ecto-5'-nucleotidase/CD73 may reduce neuronal excitability, thus providing a novel therapeutic target for drug-refractory seizures in MTLE patients.

  8. Converging translational evidence for the involvement of the serotonin 2A receptor gene in major depressive disorder.

    PubMed

    Petit, Anne-Cécile; Quesseveur, Gaël; Gressier, Florence; Colle, Romain; David, Denis J; Gardier, Alain M; Ferreri, Florian; Lépine, Jean-Pierre; Falissard, Bruno; Verstuyft, Céline; Guiard, Bruno P; Corruble, Emmanuelle

    2014-10-03

    An association between serotonin 2A receptor (5-HT2AR), encoded by HTR2A gene, and major depressive disorder (MDD) has been suggested. Here, we combined preclinical and ecological clinical approaches to explore the impact of impaired 5-HT2AR-mediated transmission on MDD or anxio-depressive-like phenotype in mice. Htr2a knock-out mice (Htr2a(-/-)) and wild-type mice were compared for the ability of chronic corticosterone to elicit some anxio-depressive-like phenotype in three behavioral paradigms (elevated plus maze, tail suspension test and splash test). Accordingly, two single nucleotide polymorphisms of the HTR2A gene (rs6314 ie His452Tyr and rs6313 ie 102C/T), which specific allelic variants may decrease 5-HT2AR-mediated transmission (as in Htr2a(-/-)mice), were studied in a sample of 485 Caucasian patients with MDD. In response to chronic corticosterone exposure, Htr2a(-/-) mice displayed more pronounced anxiodepressive-like phenotype than wild-type mice, as shown by a significant higher "emotionality score" (p<0.01). In patients, the C allele of rs6313 was more frequent in depressed patients (p=0.019) and was also associated with a more severe major depressive episode (p=0.03). This translational and ecological study involving constitutive Htr2a(-/-) knock-out mice and related SNPs in depressed patients suggests that a lower neurotransmission at the 5-HT2AR may favor the susceptibility and severity of MDE. It also suggests that specific allelic variants of the rs6313 and rs6314 may reduce 5-HT2AR-mediated transmission.

  9. Serotonin receptor gene (HTR2A) T102C polymorphism modulates individuals’ perspective taking ability and autistic-like traits

    PubMed Central

    Gong, Pingyuan; Liu, Jinting; Blue, Philip R.; Li, She; Zhou, Xiaolin

    2015-01-01

    Previous studies have indicated that empathic traits, such as perspective taking, are associated with the levels of serotonin in the brain and with autism spectrum conditions. Inspired by the finding that the serotonin receptor 2A gene (HTR2A) modulates the availability of serotonin, this study investigated to what extent HTR2A modulates individuals’ perspective taking ability and autistic-like traits. To examine the associations of the functional HTR2A polymorphism T102C (rs6313) with individuals’ perspective taking abilities and autistic-like traits, we differentiated individuals according to this polymorphism and measured empathic and autistic-like traits with Interpersonal Reactivity Index (IRI) and Autism-Spectrum Quotient (AQ) scale in 523 Chinese people. The results indicated that this polymorphism was significantly associated with the scores on Perspective Taking and Personal Distress subscales of IRI, and Communication subscale of AQ. Individuals with a greater number of the C alleles were less likely to spontaneously adopt the point of view of others, more likely to be anxious when observing the pain endured by others, and more likely to have communication problems. Moreover, the genotype effect on communication problems was mediated by individuals’ perspective taking ability. These findings provide evidence that the HTR2A T102C polymorphism is a predictor of individual differences in empathic and autistic-like traits and highlight the role of the gene in the connection between perspective taking and autistic-like traits. PMID:26557070

  10. Serotonin receptor gene (HTR2A) T102C polymorphism modulates individuals' perspective taking ability and autistic-like traits.

    PubMed

    Gong, Pingyuan; Liu, Jinting; Blue, Philip R; Li, She; Zhou, Xiaolin

    2015-01-01

    Previous studies have indicated that empathic traits, such as perspective taking, are associated with the levels of serotonin in the brain and with autism spectrum conditions. Inspired by the finding that the serotonin receptor 2A gene (HTR2A) modulates the availability of serotonin, this study investigated to what extent HTR2A modulates individuals' perspective taking ability and autistic-like traits. To examine the associations of the functional HTR2A polymorphism T102C (rs6313) with individuals' perspective taking abilities and autistic-like traits, we differentiated individuals according to this polymorphism and measured empathic and autistic-like traits with Interpersonal Reactivity Index (IRI) and Autism-Spectrum Quotient (AQ) scale in 523 Chinese people. The results indicated that this polymorphism was significantly associated with the scores on Perspective Taking and Personal Distress subscales of IRI, and Communication subscale of AQ. Individuals with a greater number of the C alleles were less likely to spontaneously adopt the point of view of others, more likely to be anxious when observing the pain endured by others, and more likely to have communication problems. Moreover, the genotype effect on communication problems was mediated by individuals' perspective taking ability. These findings provide evidence that the HTR2A T102C polymorphism is a predictor of individual differences in empathic and autistic-like traits and highlight the role of the gene in the connection between perspective taking and autistic-like traits.

  11. Effect of serotonin receptor 2A gene polymorphism on mirtazapine response in major depression.

    PubMed

    Kang, Rhee-Hun; Choi, Myoung-Jin; Paik, Jong-Woo; Hahn, Sang-Woo; Lee, Min-Soo

    2007-01-01

    The 5-HTR2A gene is a candidate gene for influencing the clinical response to treatment with antidepressants. The purpose of this study was to determine the relationship between the -1438A/G polymorphism of the 5-HTR2A gene and the response to mirtazapine in a Korean population with major depressive disorder. Mirtazapine was administered for eight weeks to the 101 patients who completed the study, during which we evaluated the clinical outcome using repeated-measures ANCOVA. A main effect of genotype or an effect of genotype-time interactions on the decrease in HAMD score during the eight-week follow-up was not found, which suggests that the 5-HTR2A -1438A/G polymorphism does not affect the clinical outcome to mirtazapine administration. However, significant effects of genotype and allele carriers on the decrease in the sleep score over the eight weeks were found (genotype: F = 4.093, p = 0.017; allele: F = 4.371, p = 0.037), whereas no effect of genotype-time interactions on the decrease in the HAMD score over the eight-week follow-up was found. These observations suggest that the -1438A/G polymorphism on the sleep improvement at each time period revealed significant differences in the sleep scores after two weeks of mirtazapine administration. The sleep scores were lower for carriers of the A+ allele than of the A- allele after two weeks of mirtazapine administration (p = 0.041), which means that the -1438GG genotype is associated with less improvement in sleep, and suggests that the effect of mirtazapine on improving the sleep quality differs with the 5-HTR2A -1438A/G polymorphism within two weeks of mirtazapine treatment. In conclusion, although the -1438A/G polymorphism affects the sleep improvement resulting from the administration of mirtazapine to Korean patients with major depressive disorder, our results do not support the hypothesis that this polymorphism of the 5-HTR2A gene is involved in the therapeutic response to mirtazapine.

  12. A2a and a2b adenosine receptors affect HIF-1α signaling in activated primary microglial cells.

    PubMed

    Merighi, Stefania; Borea, Pier Andrea; Stefanelli, Angela; Bencivenni, Serena; Castillo, Carlos Alberto; Varani, Katia; Gessi, Stefania

    2015-05-15

    Microglia are central nervous system (CNS)-resident immune cells, that play a crucial role in neuroinflammation. Hypoxia-inducible factor-1 (HIF-1), the main transcription factor of hypoxia-inducible genes, is also involved in the immune response, being regulated in normoxia by inflammatory mediators. Adenosine is an ubiquitous nucleoside that has an influence on many immune properties of microglia through interaction with four receptor subtypes. The aim of this study was to investigate whether adenosine may affect microglia functions by acting on HIF-1α modulation. Primary murine microglia were activated with lipopolysaccharide (LPS) with or without adenosine, adenosine receptor agonists and antagonists and HIF-1α accumulation and downstream genes regulation were determined. Adenosine increased LPS-induced HIF-1α accumulation leading to an increase in HIF-1α target genes involved in cell metabolism [glucose transporter-1 (GLUT-1)] and pathogens killing [inducible nitric-oxide synthase (iNOS)] but did not induce HIF-1α dependent genes related to angiogenesis [vascular endothelial growth factor (VEGF)] and inflammation [tumor necrosis factor-α (TNF-α)]. The stimulatory effect of adenosine on HIF-1α and its target genes was essentially exerted by activation of A2A through p44/42 and A2B subtypes via p38 mitogen-activated protein kinases (MAPKs) and Akt phosphorylation. Furthermore the nucleoside raised VEGF and decreased TNF-α levels, by activating A2B subtypes. In conclusion adenosine increases GLUT-1 and iNOS gene expression in a HIF-1α-dependent way, through A2A and A2B receptors, suggesting their role in the regulation of microglial cells function following injury. However, inhibition of TNF-α adds an important anti-inflammatory effect only for the A2B subtype. GLIA 2015.

  13. Role of adenosine A2A receptor signaling in the nicotine-evoked attenuation of reflex cardiac sympathetic control.

    PubMed

    El-Mas, Mahmoud M; El-Gowilly, Sahar M; Fouda, Mohamed A; Saad, Evan I

    2011-08-01

    Baroreflex dysfunction contributes to increased cardiovascular risk in cigarette smokers. Given the importance of adenosinergic pathways in baroreflex control, the hypothesis was tested that defective central adenosinergic modulation of cardiac autonomic activity mediates the nicotine-baroreflex interaction. Baroreflex curves relating changes in heart rate (HR) to increases or decreases in blood pressure (BP) evoked by i.v. doses (1-16μg/kg) of phenylephrine (PE) and sodium nitroprusside (SNP), respectively, were constructed in conscious rats; slopes of the curves were taken as measures of baroreflex sensitivity (BRS). Nicotine (25 and 100μg/kg i.v.) dose-dependently reduced BRS(SNP) in contrast to no effect on BRS(PE). BRS(SNP) was also attenuated after intracisternal (i.c.) administration of nicotine. Similar reductions in BRS(SNP) were observed in rats pretreated with atropine or propranolol. The combined treatment with nicotine and atropine produced additive inhibitory effects on BRS, an effect that was not demonstrated upon concurrent exposure to nicotine and propranolol. BRS(SNP) was reduced in preparations treated with i.c. 8-phenyltheophylline (8-PT, nonselective adenosine receptor antagonist), 8-(3-Chlorostyryl) caffeine (CSC, A(2A) antagonist), or VUF5574 (A(3) antagonist). In contrast, BRS(SNP) was preserved after blockade of A(1) (DPCPX) or A(2B) (alloxazine) receptors or inhibition of adenosine uptake by dipyridamole. CSC or 8-PT abrogated the BRS(SNP) depressant effect of nicotine whereas other adenosinergic antagonists were without effect. Together, nicotine preferentially impairs reflex tachycardia via disruption of adenosine A(2A) receptor-mediated facilitation of reflex cardiac sympathoexcitation. Clinically, the attenuation by nicotine of compensatory sympathoexcitation may be detrimental in conditions such as hypothalamic defense response, posture changes, and ventricular rhythms.

  14. Serotonin 2A Receptors, Citalopram and Tryptophan-Depletion: a Multimodal Imaging Study of their Interactions During Response Inhibition

    PubMed Central

    Macoveanu, Julian; Hornboll, Bettina; Elliott, Rebecca; Erritzoe, David; Paulson, Olaf B; Siebner, Hartwig; Knudsen, Gitte M; Rowe, James B

    2013-01-01

    Poor behavioral inhibition is a common feature of neurological and psychiatric disorders. Successful inhibition of a prepotent response in ‘NoGo' paradigms requires the integrity of both the inferior frontal gyrus (IFG) and the serotonergic system. We investigated individual differences in serotonergic regulation of response inhibition. In 24 healthy adults, we used 18F-altanserin positron emission tomography to assess cerebral 5-HT2A receptors, which have been related to impulsivity. We then investigated the impact of two acute manipulations of brain serotonin levels on behavioral and neural correlates of inhibition using intravenous citalopram and acute tryptophan depletion during functional magnetic resonance imaging. We adapted the NoGo paradigm to isolate effects on inhibition per se as opposed to other aspects of the NoGo paradigm. Successful NoGo inhibition was associated with greater activation of the right IFG compared to control trials with alternative responses, indicating that the IFG is activated with inhibition in NoGo trials rather than other aspects of invoked cognitive control. Activation of the left IFG during NoGo trials was greater with citalopram than acute tryptophan depletion. Moreover, with the NoGo-type of response inhibition, the right IFG displayed an interaction between the type of serotonergic challenge and neocortical 5-HT2A receptor binding. Specifically, acute tryptophan depletion (ATD) produced a relatively larger NoGo response in the right IFG in subjects with low 5-HT2A BPP but reduced the NoGo response in those with high 5-HT2A BPP. These links between serotonergic function and response inhibition in healthy subjects may help to interpret serotonergic abnormalities underlying impulsivity in neuropsychiatric disorders. PMID:23303045

  15. Using psilocybin to investigate the relationship between attention, working memory, and the serotonin 1A and 2A receptors.

    PubMed

    Carter, Olivia L; Burr, David C; Pettigrew, John D; Wallis, Guy M; Hasler, Felix; Vollenweider, Franz X

    2005-10-01

    Increasing evidence suggests a link between attention, working memory, serotonin (5-HT), and prefrontal cortex activity. In an attempt to tease out the relationship between these elements, this study tested the effects of the hallucinogenic mixed 5-HT1A/2A receptor agonist psilocybin alone and after pretreatment with the 5-HT2A antagonist ketanserin. Eight healthy human volunteers were tested on a multiple-object tracking task and spatial working memory task under the four conditions: placebo, psilocybin (215 microg/kg), ketanserin (50 mg), and psilocybin and ketanserin. Psilocybin significantly reduced attentional tracking ability, but had no significant effect on spatial working memory, suggesting a functional dissociation between the two tasks. Pretreatment with ketanserin did not attenuate the effect of psilocybin on attentional performance, suggesting a primary involvement of the 5-HT1A receptor in the observed deficit. Based on physiological and pharmacological data, we speculate that this impaired attentional performance may reflect a reduced ability to suppress or ignore distracting stimuli rather than reduced attentional capacity. The clinical relevance of these results is also discussed.

  16. Amelioration of hypoxia-induced striatal 5-HT(2A) receptor, 5-HT transporter and HIF1 alterations by glucose, oxygen and epinephrine in neonatal rats.

    PubMed

    Anju, T R; Paulose, C S

    2011-09-20

    Alterations in neurotransmitters and its receptors expression induce brain injury during neonatal hypoxic insult. Molecular processes regulating the serotonergic receptors play an important role in the control of respiration under hypoxic insult. The present study focused on the serotonergic regulation of neonatal hypoxia and its resuscitation methods. Receptor binding assays and gene expression studies were done to evaluate the changes in 5HT(2A) receptors and its transporter in the corpus striatum of hypoxic neonatal rats and hypoxic rats resuscitated with glucose, oxygen and epinephrine. Total 5HT and 5HT(2A) receptor number was increased in hypoxic neonates along with an up regulation of 5HT(2A) receptor and 5HT transporter gene. The enhanced striatal 5HT(2A) receptors modulate the ventilatory response to hypoxia. Immediate glucose resuscitation was found to ameliorate the receptor and transporter alterations. Hypoxia induced ATP depletion mediated reduction in blood glucose levels can be encountered by glucose administration and oxygenation helps in overcoming the anaerobic condition. The adverse effect of immediate oxygenation and epinephrine supplementation was also reported. This has immense clinical significance in establishing a proper resuscitation for the management of neonatal hypoxia.

  17. Crucial role of the 5-HT2C receptor, but not of the 5-HT2A receptor, in the down regulation of stimulated dopamine release produced by pressure exposure in freely moving rats.

    PubMed

    Kriem, B; Rostain, J C; Abraini, J H

    1998-06-15

    Helium pressure of more than 2 MPa is a well known factor underlying pressure-dependent central neuroexcitatory disorders, referred to as the high-pressure neurological syndrome. This includes an increase in both serotonin (5-HT) and dopamine (DA) release. The relationship between the increase in 5-HT transmission produced by helium pressure and its effect on DA release has been clarified in a recent study, which have first demonstrated that the helium pressure-induced increase in DA release was dependent on some 5-HT receptor activation. In the present study, we examined in freely moving rats the role of 5-HT2A and 5-HT2C receptors in the increase in DA release induced by 8 MPa helium pressure. We used the 5-HT2A receptor antagonist ketanserin and the 5-HT2C receptor agonist m-CPP which have been demonstrated to reduce DA function. Because neither ketanserin is an ideal 5-HT2A receptor antagonist nor m-CPP an ideal 5-HT2C receptor agonist, additional experiments were made at normal pressure to check up on the selectivity of ketanserin and m-CPP for 5-HT2A and 5-HT2C receptors, respectively. Administration of m-CPP reduced both DA basal level and the helium pressure-induced increase in DA release, whereas administration of ketanserin only showed a little effect on the increase in DA release produced by high helium pressure. These results suggest that the 5-HT2C receptor, but not the 5-HT2A receptor, would play a crucial role in the helium pressure-induced increase in DA release. This further suggests that helium pressure may simultaneously induce an increase in 5-HT transmission at the level of 5-HT2A receptors and a decrease in 5-HT transmission at the level of 5-HT2C receptors.

  18. Modulation of Ca2+-currents by sequential and simultaneous activation of adenosine A1 and A 2A receptors in striatal projection neurons.

    PubMed

    Hernández-González, O; Hernández-Flores, T; Prieto, G A; Pérez-Burgos, A; Arias-García, M A; Galarraga, E; Bargas, J

    2014-01-01

    D(1)- and D(2)-types of dopamine receptors are located separately in direct and indirect pathway striatal projection neurons (dSPNs and iSPNs). In comparison, adenosine A(1)-type receptors are located in both neuron classes, and adenosine A(2A)-type receptors show a preferential expression in iSPNs. Due to their importance for neuronal excitability, Ca(2+)-currents have been used as final effectors to see the function of signaling cascades associated with different G protein-coupled receptors. For example, among many other actions, D(1)-type receptors increase, while D(2)-type receptors decrease neuronal excitability by either enhancing or reducing, respectively, CaV1 Ca(2+)-currents. These actions occur separately in dSPNs and iSPNs. In the case of purinergic signaling, the actions of A(1)- and A(2A)-receptors have not been compared observing their actions on Ca(2+)-channels of SPNs as final effectors. Our hypotheses are that modulation of Ca(2+)-currents by A(1)-receptors occurs in both dSPNs and iSPNs. In contrast, iSPNs would exhibit modulation by both A(1)- and A2A-receptors. We demonstrate that A(1)-type receptors reduced Ca(2+)-currents in all SPNs tested. However, A(2A)-type receptors enhanced Ca(2+)-currents only in half tested neurons. Intriguingly, to observe the actions of A(2A)-type receptors, occupation of A(1)-type receptors had to occur first. However, A(1)-receptors decreased Ca(V)2 Ca(2+)-currents, while A(2A)-type receptors enhanced current through Ca(V)1 channels. Because these channels have opposing actions on cell discharge, these differences explain in part why iSPNs may be more excitable than dSPNs. It is demonstrated that intrinsic voltage-gated currents expressed in SPNs are effectors of purinergic signaling that therefore play a role in excitability.

  19. Interferon-α Induces Neurotoxicity Through Activation of the Type I Receptor and the GluN2A Subunit of the NMDA Receptor

    PubMed Central

    Kessing, Cari F.

    2015-01-01

    Elevated levels of interferon-alpha (IFNα) in the central nervous system (CNS) are linked to cognitive dysfunction in patients with inflammatory CNS diseases such as HIV-associated neurocognitive disorders (HAND). Increased CNS IFNα has also been found to be associated with cognitive dysfunction in a HAND mouse model. Here, we corroborate previous studies showing a dose-dependent decrease in dendritic branching and length caused by IFNα treatment and extend those studies. Because both direct and indirect mechanisms of IFNα-induced neurotoxicity are likely involved, the cell signaling pathway involving the IFNα receptor (IFNAR) was initially evaluated. Rat neuronal cultures exposed to IFNα demonstrate increased phosphorylation of STAT1 and increased interferon stimulating gene 15 (ISG15) expression, indicators of IFNAR engagement. However, specific blocking antibodies to the IFNAR were found to only partially protect neurons from IFNα-induced neurotoxicity. Additionally, inhibiting the GluN2A subunit of N-methyl-D-asparate receptor (NMDAR) was also found to be partially protective against IFNα-induced neurotoxicity compared with the GluN2B subunit. Neurotoxicity is evident in neurons extracted from IFNAR KO mice treated with IFNα as well, further indicating that IFNAR signaling is not required for IFNα neurotoxicity. The neurotoxic actions of IFNα are mediated through both the IFNAR as well as the GluN2A subunit of the NMDAR to reduce dendritic arborization in neurons. Complete protection from IFNα-induced neurotoxicity was demonstrated when both pathways were blocked. Blocking these pathways could lead to potential therapies for cognitive dysfunction during neuroinflammation and specifically lead to better treatments for HAND. PMID:25517826

  20. Structure and variation of three canine genes involved in serotonin binding and transport: the serotonin receptor 1A gene (htr1A), serotonin receptor 2A gene (htr2A), and serotonin transporter gene (slc6A4).

    PubMed

    van den Berg, L; Kwant, L; Hestand, M S; van Oost, B A; Leegwater, P A J

    2005-01-01

    Aggressive behavior is the most frequently encountered behavioral problem in dogs. Abnormalities in brain serotonin metabolism have been described in aggressive dogs. We studied canine serotonergic genes to investigate genetic factors underlying canine aggression. Here, we describe the characterization of three genes of the canine serotonergic system: the serotonin receptor 1A and 2A gene (htr1A and htr2A) and the serotonin transporter gene (slc6A4). We isolated canine bacterial artificial chromosome clones containing these genes and designed oligonucleotides for genomic sequencing of coding regions and intron-exon boundaries. Golden retrievers were analyzed for DNA sequence variations. We found two nonsynonymous single nucleotide polymorphisms (SNPs) in the coding sequence of htr1A; one SNP close to a splice site in htr2A; and two SNPs in slc6A4, one in the coding sequence and one close to a splice site. In addition, we identified a polymorphic microsatellite marker for each gene. Htr1A is a strong candidate for involvement in the domestication of the dog. We genotyped the htr1A SNPs in 41 dogs of seven breeds with diverse behavioral characteristics. At least three SNP haplotypes were found. Our results do not support involvement of the gene in domestication.

  1. Targeted mutagenesis of aryl hydrocarbon receptor 2a and 2b genes in Atlantic killifish (Fundulus heteroclitus).

    PubMed

    Aluru, Neelakanteswar; Karchner, Sibel I; Franks, Diana G; Nacci, Diane; Champlin, Denise; Hahn, Mark E

    2015-01-01

    Understanding molecular mechanisms of toxicity is facilitated by experimental manipulations, such as disruption of function by gene targeting, that are especially challenging in non-standard model species with limited genomic resources. While loss-of-function approaches have included gene knock-down using morpholino-modified oligonucleotides and random mutagenesis using mutagens or retroviruses, more recent approaches include targeted mutagenesis using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. These latter methods provide more accessible opportunities to explore gene function in non-traditional model species. To facilitate evaluation of toxic mechanisms for important categories of aryl hydrocarbon pollutants, whose actions are known to be receptor mediated, we used ZFN and CRISPR-Cas9 approaches to generate aryl hydrocarbon receptor 2a (AHR2a) and AHR2b gene mutations in Atlantic killifish (Fundulus heteroclitus) embryos. This killifish is a particularly valuable non-traditional model, with multiple paralogs of AHR whose functions are not well characterized. In addition, some populations of this species have evolved resistance to toxicants such as halogenated aromatic hydrocarbons. AHR-null killifish will be valuable for characterizing the role of the individual AHR paralogs in evolved resistance, as well as in normal development. We first used five-finger ZFNs targeting exons 1 and 3 of AHR2a. Subsequently, CRISPR-Cas9 guide RNAs were designed to target regions in exon 2 and 3 of AHR2a and AHR2b. We successfully induced frameshift mutations in AHR2a exon 3 with ZFN and CRISPR-Cas9 guide RNAs, with mutation frequencies of 10% and 16%, respectively. In AHR2b, mutations were induced using CRISPR-Cas9 guide RNAs targeting sites in both exon 2 (17%) and exon 3 (63%). We screened AHR2b exon 2 CRISPR-Cas9-injected embryos for off

  2. Selected C8 two-chain linkers enhance the adenosine A1/A2A receptor affinity and selectivity of caffeine.

    PubMed

    van der Walt, M M; Terre'Blanche, G

    2017-01-05

    Recent research exploring C8 substitution on the caffeine core identified 8-(2-phenylethyl)-1,3,7-trimethylxanthine as a non-selective adenosine receptor antagonist. To elaborate further, we included various C8 two-chain-length linkers to enhance adenosine receptor affinity. The results indicated that the unsubstituted benzyloxy linker (1e A1Ki = 1.52 μM) displayed the highest affinity for the A1 adenosine receptor and the para-chloro-substituted phenoxymethyl (1d A2AKi = 1.33 μM) linker the best A2A adenosine receptor affinity. The position of the oxygen revealed that the phenoxymethyl linker favoured A1 adenosine receptor selectivity over the benzyloxy linker and, by introducing a para-chloro substituent, A2A adenosine receptor selectivity was obtained. Selected compounds (1c, 1e) behaved as A1 adenosine receptor antagonists in GTP shift assays and therefore represent selective and non-selective A1 and A2A adenosine receptor antagonists that may have potential for treating neurological disorders.

  3. E6-Associated Protein Dependent Estrogen Receptor Regulation of Protein Kinase A Regulatory Subunit R2A Expression in Neuroblastoma.

    PubMed

    Obeid, Jean-Pierre; Zeidan, Youssef H; Zafar, Nawal; El Hokayem, Jimmy

    2017-02-18

    E6ap is a known transcriptional coregulator for estrogen receptor alpha (Er, Erα) in the presence of estrogen. Protein kinase A (PKA) contains two regulatory subunits derived from four genes. Recent evidence demonstrates that PKA regulates E6ap activity. Data generated in our lab indicated estrogen dependent regulation of Pkar2a levels. Our project sets to investigate a possible feedback mechanism constituting of Erα and E6ap transcriptional regulation of Pkar2a expression. Western blot evaluated protein regulation correlations with E2 in mouse neuroblastoma lines. Bioinformatics detected estrogen response element (ERE) sequences. quantitative polymerase chain reaction (qPCR) validated the western blot results. ERE oligonucleotides were synthesized. Reporter gene transcriptional activity was evaluated via Luciferase assay output. Electromobility shift assay (EMSA) assessed direct binding between Erα relevant sequences. Chromatin immunoprecipitation (ChIP) and Re-ChIP were conducted in quantifying protein complex recruitment levels. Pkar2a protein expression directly correlated with E2, and four putative ERE sequences were identified. Pkar2a mRNA expression reverted to baseline with either E2 or E6ap absent. In the presence of E2, ERE-1 and ERE-4 possessed Luciferase reporter gene transcriptional capabilities. ERE-1 portrayed band shifts, representing direct binding to Erα with E2 supplementation. With E2, ERE-1 significantly enhanced Erα and E6ap recruitment levels to the Pkar2a promoter. Pkar2a is directly regulated by Erα and E6ap in the presence of estrogen stimulus. This work indicates a feedback mechanism in the interplay between PKA and E6ap, which may prove crucial for the role of both proteins in cancers and neurogenetic diseases like Angelman syndrome.

  4. Role of 5-HT1B, 5-HT2A and 5-HT2C receptors in learning.

    PubMed

    Meneses, A; Hong, E

    1997-08-01

    The effects of post-training (i.p.) injection of TFMPP, mCPP, DOI or 1-NP in the autoshaping learning task was explored. Furthermore, the post-training effects of these agonists after treatment with the antagonists (+/-)-pindolol, (+/-)-propranolol, NAN-190, ketanserin, ritanserin, mesulergine, MDL-72222 or p-chloroamphetamine (5-HT depleter) were studied. Rats were individually trained with a lever-press response (conditioned response; CR) on the autoshaping task and tested 24 h later. The results showed that the injection of TFMPP (1-10 mg/kg), mCPP (1-10 mg/kg), 1-NP (0.1-1.0 mg/kg) or mesulergine (0.4 mg/kg) decreased the rate of CR, while DOI (0.01-0.1 mg/kg) and ritanserin (0.5 mg/kg) and ketanserin (0.001-0.1 mg/kg) increased it. However, the effect induced by TFMPP was reversed by (+/-)-pindolol, ketanserin, ritanserin and PCA; the mCPP-induced effect was antagonized by (+/-)-propranolol, ketanserin, ritanserin and MDL-72222; and the effect produced by 1-NP was reversed by ketanserin, ritanserin and PCA. In addition, the increment in CR provoked by DOI was enhanced by ketanserin, and reversed by ritanserin, mesulergine and PCA. These findings suggest that TFMPP, 1-NP and DOI exerted their effects via stimulation of presynaptic 5-HT receptors. The effects of mCPP most probably reflect activation of postsynaptic receptors. The present data suggest that both 5-HT1B and 5-HT2A-2C receptors play a significant role in the consolidation of learning.

  5. Stimulation of postsynapse adrenergic α2A receptor improves attention/cognition performance in an animal model of attention deficit hyperactivity disorder.

    PubMed

    Kawaura, Kazuaki; Karasawa, Jun-ichi; Chaki, Shigeyuki; Hikichi, Hirohiko

    2014-08-15

    A 5-trial inhibitory avoidance test using spontaneously hypertensive rat (SHR) pups has been used as an animal model of attention deficit hyperactivity disorder (ADHD). However, the roles of noradrenergic systems, which are involved in the pathophysiology of ADHD, have not been investigated in this model. In the present study, the effects of adrenergic α2 receptor stimulation, which has been an effective treatment for ADHD, on attention/cognition performance were investigated in this model. Moreover, neuronal mechanisms mediated through adrenergic α2 receptors were investigated. We evaluated the effects of both clonidine, a non-selective adrenergic α2 receptor agonist, and guanfacine, a selective adrenergic α2A receptor agonist, using a 5-trial inhibitory avoidance test with SHR pups. Juvenile SHR exhibited a shorter transfer latency, compared with juvenile Wistar Kyoto (WKY) rats. Both clonidine and guanfacine significantly prolonged the transfer latency of juvenile SHR. The effects of clonidine and guanfacine were significantly blocked by pretreatment with an adrenergic α2A receptor antagonist. In contrast, the effect of clonidine was not attenuated by pretreatment with an adrenergic α2B receptor antagonist, or an adrenergic α2C receptor antagonist, while it was attenuated by a non-selective adrenergic α2 receptor antagonist. Furthermore, the effects of neither clonidine nor guanfacine were blocked by pretreatment with a selective noradrenergic neurotoxin. These results suggest that the stimulation of the adrenergic α2A receptor improves the attention/cognition performance of juvenile SHR in the 5-trial inhibitory avoidance test and that postsynaptic, rather than presynaptic, adrenergic α2A receptor is involved in this effect.

  6. Persistent reduction of cocaine seeking by pharmacological manipulation of adenosine A1 and A2A receptors during extinction training in rats

    PubMed Central

    O’Neill, Casey E.; Hobson, Benjamin D.; Levis, Sophia C.; Bachtell, Ryan K.

    2014-01-01

    Rationale Adenosine receptor stimulation and blockade has been shown to modulate a variety of cocaine related behaviors. Objectives These studies identify the direct effects of adenosine receptor stimulation on cocaine seeking during extinction training and the persistent effects on subsequent reinstatement to cocaine seeking. Methods Rats self-administered cocaine on a fixed-ratio 1 schedule in daily sessions over 3 weeks. Following 1 week withdrawal, the direct effects of adenosine receptor modulation were tested by administering the adenosine A1 receptor agonist, CPA (0.03 mg/kg and 0.1 mg/kg), the adenosine A2A agonist, CGS 21680 (0.03 mg/kg and 0.1 mg/kg), the presynaptic adenosine A2A receptor antagonist, SCH 442416 (0.3 mg/kg, 1 mg/kg, and 3 mg/kg), or vehicle prior to each of 6 daily extinction sessions. The persistent effects of adenosine receptor modulation during extinction training were subsequently tested on reinstatement to cocaine seeking induced by cues, cocaine, and the dopamine D2 receptor agonist, quinpirole. Results All doses of CPA and CGS 21680 impaired initial extinction responding, however only CPA treatment during extinction produced persistent impairment in subsequent cocaine- and quinpirole-induced seeking. Dissociating CPA treatment from extinction did not alter extinction responding or subsequent reinstatement. Administration of SCH 442416 had no direct effects on extinction responding, but produced dose-dependent persistent impairment of cocaine- and quinpirole-induced seeking. Conclusions These findings demonstrate that adenosine A1 or A2A receptor stimulation directly impair extinction responding. Interestingly, adenosine A1 receptor stimulation or presynaptic adenosine A2A receptor blockade during extinction produces lasting changes in relapse susceptibility. PMID:24562064

  7. G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent

    PubMed Central

    Jamshad, Mohammed; Charlton, Jack; Lin, Yu-Pin; Routledge, Sarah J.; Bawa, Zharain; Knowles, Timothy J.; Overduin, Michael; Dekker, Niek; Dafforn, Tim R.; Bill, Roslyn M.; Poyner, David R.; Wheatley, Mark

    2015-01-01

    G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([3H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms. PMID:25720391

  8. Protein kinase Cε is required for spinal analgesic synergy between delta opioid and alpha-2A adrenergic receptor agonist pairs.

    PubMed

    Schuster, Daniel J; Kitto, Kelley F; Overland, Aaron C; Messing, Robert O; Stone, Laura S; Fairbanks, Carolyn A; Wilcox, George L

    2013-08-14

    We recently showed that spinal synergistic interactions between δ opioid receptors (δORs) and α2A adrenergic receptors (α2AARs) require protein kinase C (PKC). To identify which PKC isoforms contribute to analgesic synergy, we evaluated the effects of various PKC-isoform-specific peptide inhibitors on synergy between δORs and α2AARs using the tail flick assay of thermal nociception in mice. Only a PKCε inhibitor abolished synergy between a δOR agonist and an α2AAR agonist. We tested a panel of combinations of opioid and adrenergic agonists in PKCε knock-out mice and found that all four combinations of a δOR agonist and an α2AAR agonist required PKCε for antinociceptive synergy. None of the combinations of a μOR agonist with an α2AR agonist required PKCε. Immunohistochemistry confirmed that PKCε could be found in the population of peptidergic primary afferent nociceptors where δORs and α2AARs have been found to extensively colocalize. Immunoreactivity for PKCε was found in the majority of dorsal root ganglion neurons and intensely labeled laminae I and II of the spinal cord dorsal horn. PKCε is widespread in the spinal nociceptive system and in peptidergic primary afferents it appears to be specifically involved in mediating the synergistic interaction between δORs and α2AARs.

  9. Distribution of 5-HT2A receptor immunoreactivity in the rat amygdaloid complex and colocalization with γ-aminobutyric acid.

    PubMed

    Bombardi, Cristiano

    2011-01-25

    The 5-HT2A receptor (5-HT2Ar) is located in a variety of excitatory and inhibitory neurons in many regions of the central nervous system and is a major target for atypical antipsychotic drugs. In the present study, an immunoperoxidase experiment was used to investigate the distribution of 5-HT2Ar immunoreactivity in the rat amygdaloid complex. In the basolateral amygdala, the colocalization of 5-HT2Ar with inhibitory transmitter γ-aminobutyric acid (GABA) was studied using double-immunofluorescence confocal microscopy. The staining pattern obtained was colchicine-sensitive. In fact, pretreatment with colchicine increased the number of 5-HT2Ar-immunoreactive somata. Accordingly, with the exception of the intercalated nuclei, the amygdaloid complex of colchicine-injected rats exhibited a high density of 5-HT2Ar-IR somata. Morphological analyses indicated that 5-HT2Ar was located on both excitatory and inhibitory neurons in the rat amygdaloid complex. In addition, double-immunofluorescence observations revealed that the great majority of GABA-immunoreactive neurons in the basolateral amygdala exhibited 5-HT2Ar immunoreactivity (66.3%-70.6% depending on the nucleus). These data help to clarify the complex role of the 5-HT2Ar in the amygdaloid complex suggesting that this receptor can regulate amygdaloid activity by acting on different neuronal populations.

  10. Protein Kinase Cϵ Is Required for Spinal Analgesic Synergy between Delta Opioid and Alpha-2A Adrenergic Receptor Agonist Pairs

    PubMed Central

    Schuster, Daniel J.; Kitto, Kelley F.; Overland, Aaron C.; Messing, Robert O.; Stone, Laura S.; Fairbanks, Carolyn A.

    2013-01-01

    We recently showed that spinal synergistic interactions between δ opioid receptors (δORs) and α2A adrenergic receptors (α2AARs) require protein kinase C (PKC). To identify which PKC isoforms contribute to analgesic synergy, we evaluated the effects of various PKC-isoform-specific peptide inhibitors on synergy between δORs and α2AARs using the tail flick assay of thermal nociception in mice. Only a PKCϵ inhibitor abolished synergy between a δOR agonist and an α2AAR agonist. We tested a panel of combinations of opioid and adrenergic agonists in PKCϵ knock-out mice and found that all four combinations of a δOR agonist and an α2AAR agonist required PKCϵ for antinociceptive synergy. None of the combinations of a μOR agonist with an α2AR agonist required PKCϵ. Immunohistochemistry confirmed that PKCϵ could be found in the population of peptidergic primary afferent nociceptors where δORs and α2AARs have been found to extensively colocalize. Immunoreactivity for PKCϵ was found in the majority of dorsal root ganglion neurons and intensely labeled laminae I and II of the spinal cord dorsal horn. PKCϵ is widespread in the spinal nociceptive system and in peptidergic primary afferents it appears to be specifically involved in mediating the synergistic interaction between δORs and α2AARs. PMID:23946412

  11. Salidroside attenuates chronic hypoxia-induced pulmonary hypertension via adenosine A2a receptor related mitochondria-dependent apoptosis pathway.

    PubMed

    Huang, Xiaoying; Zou, Lizhen; Yu, Xiaoming; Chen, Mayun; Guo, Rui; Cai, Hui; Yao, Dan; Xu, Xiaomei; Chen, Yanfan; Ding, Cheng; Cai, Xueding; Wang, Liangxing

    2015-05-01

    Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial remodeling mainly due to excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Salidroside, an active ingredient isolated from Rhodiola rosea is proposed to exert protective effects against PAH. However, the function of salidroside in PAH has not been investigated systematically and the underlying mechanisms are not clear. To investigate the effects of salidroside on PAH, the mice in chronic hypoxia model of PAH were given by an increasing concentration of salidroside (0, 16 mg/kg, 32 mg/kg, and 64 mg/kg). After salidroside treatment, the chronic hypoxia-induced right ventricular hypertrophy and pulmonary arterial remodeling were attenuated, suggesting a protective role played by salidroside in PAH. To explore the potential mechanisms, the apoptosis of PASMCs after salidroside treatment under hypoxia conditions were determined in vivo and in vitro, and also the mitochondria-dependent apoptosis factors, Bax, Bcl-2, cytochrome C, and caspase 9 were examined. The results revealed that salidroside reversed hypoxia-induced cell apoptosis resistance at least partially via a mitochondria-dependent pathway. In addition, salidroside upregulated the expression of adenosine A2a receptor (A2aR) in lung tissues of mice and in PASMCs in vitro after hypoxia exposure. Combined the evidence above, we conclude that salidroside can attenuate chronic hypoxia-induced PAH by promoting PASMCs apoptosis via an A2aR related mitochondria dependent pathway.

  12. A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

    PubMed Central

    Kim, Do-Geun; Bynoe, Margaret S.

    2016-01-01

    The blood-brain barrier (BBB) protects the brain from toxic substances within the peripheral circulation. It maintains brain homeostasis and is a hurdle for drug delivery to the CNS to treat neurodegenerative diseases, including Alzheimer’s disease and brain tumors. The drug efflux transporter P-glycoprotein (P-gp) is highly expressed on brain endothelial cells and blocks the entry of most drugs delivered to the brain. Here, we show that activation of the A2A adenosine receptor (AR) with an FDA-approved A2A AR agonist (Lexiscan) rapidly and potently decreased P-gp expression and function in a time-dependent and reversible manner. We demonstrate that downmodulation of P-gp expression and function coincided with chemotherapeutic drug accumulation in brains of WT mice and in primary mouse and human brain endothelial cells, which serve as in vitro BBB models. Lexiscan also potently downregulated the expression of BCRP1, an efflux transporter that is highly expressed in the CNS vasculature and other tissues. Finally, we determined that multiple pathways, including MMP9 cleavage and ubiquitinylation, mediated P-gp downmodulation. Based on these data, we propose that A2A AR activation on BBB endothelial cells offers a therapeutic window that can be fine-tuned for drug delivery to the brain and has potential as a CNS drug-delivery technology. PMID:27043281

  13. Somatostatin and its 2A receptor in dorsal root ganglia and dorsal horn of mouse and human: expression, trafficking and possible role in pain

    PubMed Central

    2014-01-01

    Background Somatostatin (SST) and some of its receptor subtypes have been implicated in pain signaling at the spinal level. In this study we have investigated the role of SST and its sst2A receptor (sst2A) in dorsal root ganglia (DRGs) and spinal cord. Results SST and sst2A protein and sst2 transcript were found in both mouse and human DRGs, sst2A-immunoreactive (IR) cell bodies and processes in lamina II in mouse and human spinal dorsal horn, and sst2A-IR nerve terminals in mouse skin. The receptor protein was associated with the cell membrane. Following peripheral nerve injury sst2A-like immunoreactivity (LI) was decreased, and SST-LI increased in DRGs. sst2A-LI accumulated on the proximal and, more strongly, on the distal side of a sciatic nerve ligation. Fluorescence-labeled SST administered to a hind paw was internalized and retrogradely transported, indicating that a SST-sst2A complex may represent a retrograde signal. Internalization of sst2A was seen in DRG neurons after systemic treatment with the sst2 agonist octreotide (Oct), and in dorsal horn and DRG neurons after intrathecal administration. Some DRG neurons co-expressed sst2A and the neuropeptide Y Y1 receptor on the cell membrane, and systemic Oct caused co-internalization, hypothetically a sign of receptor heterodimerization. Oct treatment attenuated the reduction of pain threshold in a neuropathic pain model, in parallel suppressing the activation of p38 MAPK in the DRGs Conclusions The findings highlight a significant and complex role of the SST system in pain signaling. The fact that the sst2A system is found also in human DRGs and spinal cord, suggests that sst2A may represent a potential pharmacologic target for treatment of neuropathic pain. PMID:24521084

  14. Effects of the 5-HT receptor antagonists GR127935 (5-HT1B/1D) and MDL100907 (5-HT2A) in the consolidation of learning.

    PubMed

    Meneses, A; Terrón, J A; Hong, E

    1997-12-01

    We have previously reported that 5-HT1B/1D and 5-HT2A/2B/2C receptors play a role in learning and memory. The present investigation was devoted to analyze further in the autoshaping learning task: (1) the effects of the 5-HT1A/1B/1D receptor agonist, GR46611, the 5-HT1B/1D receptor antagonist, GR127935, and the selective 5-HT2A receptor antagonist, MDL100907. Consistent with a role of 5-HT1B/1D receptors in learning, the post-training injection of GR46611 (1-10 mg/kg) decreased the consolidation of learning whereas GR127935 (10 mg/kg) increased it; the effects of both drugs were reversed by PCA pretreatment. GR127935 abolished the decrease induced by GR46611, TFMPP and mCPP, whereas MDL100907 (0.1-3.0 mg/kg) had no effect by itself but abolished the effects of DOI, ketanserin and TFMPP and moderately inhibited the effects elicited by mCPP, 1-NP and mesulergine. Neither did GR127935 nor MDL100907 significantly modify the increase in the consolidation of learning induced by 8-OH-DPAT. Thus, the present findings suggest that stimulation of presynaptic 5-HT1B/1D receptors impairs the consolidation of learning whilst stimulation of 5-HT2A/2C receptors enhances it; the blockade of 5-HT2A receptors has no effects. In addition, 5-HT2 receptors seem to modulate this cognitive stage.

  15. Activation of serotonin 2A receptors underlies the psilocybin-induced effects on α oscillations, N170 visual-evoked potentials, and visual hallucinations.

    PubMed

    Kometer, Michael; Schmidt, André; Jäncke, Lutz; Vollenweider, Franz X

    2013-06-19

    Visual illusions and hallucinations are hallmarks of serotonergic hallucinogen-induced altered states of consciousness. Although the serotonergic hallucinogen psilocybin activates multiple serotonin (5-HT) receptors, recent evidence suggests that activation of 5-HT2A receptors may lead to the formation of visual hallucinations by increasing cortical excitability and altering visual-evoked cortical responses. To address this hypothesis, we assessed the effects of psilocybin (215 μg/kg vs placebo) on both α oscillations that regulate cortical excitability and early visual-evoked P1 and N170 potentials in healthy human subjects. To further disentangle the specific contributions of 5-HT2A receptors, subjects were additionally pretreated with the preferential 5-HT2A receptor antagonist ketanserin (50 mg vs placebo). We found that psilocybin strongly decreased prestimulus parieto-occipital α power values, thus precluding a subsequent stimulus-induced α power decrease. Furthermore, psilocybin strongly decreased N170 potentials associated with the appearance of visual perceptual alterations, including visual hallucinations. All of these effects were blocked by pretreatment with the 5-HT2A antagonist ketanserin, indicating that activation of 5-HT2A receptors by psilocybin profoundly modulates the neurophysiological and phenomenological indices of visual processing. Specifically, activation of 5-HT2A receptors may induce a processing mode in which stimulus-driven cortical excitation is overwhelmed by spontaneous neuronal excitation through the modulation of α oscillations. Furthermore, the observed reduction of N170 visual-evoked potentials may be a key mechanism underlying 5-HT2A receptor-mediated visual hallucinations. This change in N170 potentials may be important not only for psilocybin-induced states but also for understanding acute hallucinatory states seen in psychiatric disorders, such as schizophrenia and Parkinson's disease.

  16. Increasing spinal 5-HT2A receptor responsiveness mediates anti-allodynic effect and potentiates fluoxetine efficacy in neuropathic rats. Evidence for GABA release.

    PubMed

    Dupuis, Amandine; Wattiez, Anne-Sophie; Pinguet, Jérémy; Richard, Damien; Libert, Frédéric; Chalus, Maryse; Aissouni, Youssef; Sion, Benoit; Ardid, Denis; Marin, Philippe; Eschalier, Alain; Courteix, Christine

    2017-04-01

    Antidepressants are one of the first line treatments for neuropathic pain but their use is limited by the incidence and severity of side effects of tricyclics and the weak effectiveness of selective serotonin reuptake inhibitors (SSRIs). Serotonin type 2A (5-HT2A) receptors interact with PDZ proteins that regulate their functionality and SSRI efficacy to alleviate pain. We investigated whether an interfering peptide (TAT-2ASCV) disrupting the interaction between 5-HT2A receptors and associated PDZ proteins would improve the treatment of traumatic neuropathic allodynia. Tactile allodynia was assessed in spinal nerve ligation-induced neuropathic pain in rats using von Frey filaments after acute treatment with TAT-2ASCV and/or 5-HT2A receptor agonist, alone or in combination with repeated treatment with fluoxetine. In vivo microdialysis was performed in order to examine the involvement of GABA in TAT-2ASCV/fluoxetine treatment-associated analgesia. TAT-2ASCV (100ng, single i.t. injection) improved SNL-induced tactile allodynia by increasing 5-HT2A receptor responsiveness to endogenous 5-HT. Fluoxetine alone (10mg/kg, five i.p. injections) slightly increased tactile thresholds and its co-administration with TAT-2ASCV (100ng, single i.t. injection) further enhanced the anti-allodynic effect. This effect depends on the integrity of descending serotonergic bulbospinal pathways and spinal release of GABA. The anti-allodynic effect of fluoxetine can be enhanced by disrupting 5-HT2A receptor-PDZ protein interactions. This enhancement depends on 5-HT2A receptor activation, spinal GABA release and GABAA receptor activation.

  17. Psychological, neuroimaging, and biochemical studies on functional association between impulsive behavior and the 5-HT2A receptor gene polymorphism in humans.

    PubMed

    Nomura, Michio; Nomura, Yasuyuki

    2006-11-01

    It has been suggested that impulsive behavior is caused by dysfunctional serotonergic 5-HT neurotransmission in the central nervous system (CNS). Brain neuroimaging studies have shown that behavioral inhibition is linked to the activation of cortex sites such as the ventral frontal cortex. Positron emission tomography (PET) imaging with [(18)F]altanserin to characterize 5-HT(2A) receptor binding revealed a reduction in 5-HT(2A) binding in the ventral frontal cortex in women who had recovered from impulsive diseases. These clinical, neuroimaging, and pharmacological studies appear to support the hypothesis that functional alteration of neurotransmission due to genetic polymorphisms of the 5-HT receptors may be involved in impulsive behavior modulation. Following evaluation by a self-reporting measure, it was proposed that a polymorphism in the promoter of the 5-HT(2A) receptor gene is the underlying cause of impulsive behavior; however, this hypothesis is not convincing. We examined whether the polymorphism in the 5-HT(2A) receptor gene promoter is involved in impulsive aggression by evaluating a behavioral task (Go/No-go task) in normal volunteers. The polymorphism of the 5-HT(2A) receptor gene promoter in lymphocytes from 71 volunteers was analyzed by using PCR. Impulsivity was defined as the number of commission errors (responding when one should not) recorded during a Go/No-go task; a larger number of commission errors indicate greater difficulty in inhibiting impulsive behavior. The subjects of the A-1438A allele group for the 5-HT(2A) receptor gene made more commission errors under the punishment-reward (PR)condition in a Go/No-go task than those in the G-1438G group. In the present review, we discuss and suggest the possible involvement of the A-1438A polymorphism of the 5HT2A receptor gene promoter in impulsive behavior. This hypothesis was evaluated by using a behavioral task measure that could directly reveal impulsive behavioral traits in humans.

  18. Suppression of piriform cortex activity in rat by corticotropin-releasing factor 1 and serotonin 2A/C receptors.

    PubMed

    Narla, Chakravarthi; Dunn, Henry A; Ferguson, Stephen S G; Poulter, Michael O

    2015-01-01

    The piriform cortex (PC) is richly innervated by corticotropin-releasing factor (CRF) and serotonin (5-HT) containing axons arising from central amygdala and Raphe nucleus. CRFR1 and 5-HT2A/2CRs have been shown to interact in manner where CRFR activation subsequently potentiates the activity of 5-HT2A/2CRs. The purpose of this study was to determine how the activation of CRFR1 and/or 5-HT2Rs modulates PC activity at both the circuit and cellular level. Voltage sensitive dye imaging showed that CRF acting through CRFR1 dampened activation of the Layer II of PC and interneurons of endopiriform nucleus. Application of the selective 5-HT2A/CR agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) following CRFR1 activation potentiated this effect. Blocking the interaction between CRFR1 and 5-HT2R with a Tat-CRFR1-CT peptide abolished this potentiation. Application of forskolin did not mimic CRFR1 activity but instead blocked it, while a protein kinase A antagonist had no effect. However, activation and antagonism of protein kinase C (PKC) either mimicked or blocked CRF modulation, respectively. DOI had no effect when applied alone indicating that the prior activation of CRFR1 receptors was critical for DOI to show significant effects similar to CRF. Patch clamp recordings showed that both CRF and DOI reduced the synaptic responsiveness of Layer II pyramidal neurons. CRF had highly variable effects on interneurons within Layer III, both increasing and decreasing their excitability, but DOI had no effect on the excitability of this group of neurons. These data show that CRF and 5-HT, acting through both CRFR1 and 5-HT2A/CRs, reduce the activation of the PC. This modulation may be an important blunting mechanism of stressor behaviors mediated through the olfactory cortex.

  19. Role for NKG2-A and NKG2-C surface receptors in chronic CD4+ T-cell responses.

    PubMed

    Ortega, Consuelo; Romero, Pilar; Palma, Agustín; Orta, Teresa; Peña, José; García-Vinuesa, Amaya; Molina, Ignacio J; Santamaría, Manuel

    2004-12-01

    The participation of CD94 and NKG2 gene family members in the function of NK cells and CD8+ cytolytic cells has recently been addressed in detail. However, the role that these molecules play in the key CD4+ regulatory cells remains largely unexplored. This study has examined the expression and regulation of CD94 and NKG2 genes in purified human peripheral CD4+ cells stimulated with several agents. We found a constitutive expression of NKG2-E in CD94-depleted resting peripheral CD4+ cells, whereas inductions of NKG2-A and NKG2-C required chronic cell activation and occurred after expression of CD94. We found that CD3-mediated stimulation induces the expression of CD94 first by day 5 of culture, followed by NKG2-A by day 15 and finally NKG2-C, which is not detected until 20 days after repeated stimulation. This pattern of gene expression differs sharply from that observed in purified CD8+ T cells, where mRNA from all NKG2 gene family members are detected after 5 days of stimulation. Selective activation of TCR V beta 2-bearing cells with toxic shock syndrome toxin-1 superantigen reveals that mRNA induction of NKG2-A and NKG2-C genes is significantly influenced by the presence of cytokines (IL-10 and TGF-beta) and by the restimulation of the cells. In addition, the occupancy of the CD94/NKG2-A receptor expressed on these superantigen-stimulated CD4+ T lymphocytes abrogates TNF-alpha and IFN-gamma production, whereas NKG2-C enhances production of these cytokines. Taken together our results reveal strict gene regulatory mechanisms for CD94 and NKG2 gene expression on CD4+ cells that are different from those governing the expression of these same genes in CD8+ cells. The results suggest that these genes also participate in chronic CD4+ T-cell responses.

  20. Food Cravings, Food Addiction, and a Dopamine-Resistant (DRD2 A1) Receptor Polymorphism in Asian American College Students

    PubMed Central

    Yeh, Joanna; Trang, Amy; Henning, Susanne M; Wilhalme, Holly; Carpenter, Catherine; Heber, David; Li, Zhaoping

    2016-01-01

    Background/Objectives In an era where obesity remains an important public health concern, food addiction has emerged as a possible contributor to obesity. The DRD2 gene is the most studied polymorphism. The aim of this study was to investigate a relationship between food craving and food addiction questionnaires, body composition measurements, and a dopamine-resistant receptor polymorphism (DRD2 A1) among healthy Asian Americans. Subjects/Methods A total of 84 Asian American college students were recruited. Participants underwent body composition measurement via bioelectrical impedance, answered subjective questionnaires, and had blood drawn for genotyping. Results Among Asian American college students, there was no difference in body composition (BMI, percent body fat) between the A1 (A1A1 or A1A2) and A2 (A2A2) groups. There were statistically significant differences in food cravings of carbohydrates and fast food on the Food Craving Inventory between the A1 and A2 groups (p=0.03), but not for sugar or fat. Among female Asian college students, there was also a difference on the Power of Food questionnaire (p=0.04), which was not seen among males. 13 out of 55 females also had > 30% body fat at a BMI of 21.4 to 28.5 kg/m2. Conclusion Greater carbohydrate and fast food craving was associated with the DRD2 A1 versus A2 allele among Asian Americans. Further studies examining the ability of dopamine agonists to affect food craving and to reduce body fat in Asian American are warranted. More studies in food addiction among obese Asian Americans are needed with careful definition of obesity, specifically for Asian women. PMID:27222427

  1. Potentiation by tonic A2a-adenosine receptor activation of CGRP-facilitated [3H]-ACh release from rat motor nerve endings.

    PubMed Central

    Correia-de-Sá, P.; Ribeiro, J. A.

    1994-01-01

    1. The effect of calcitonin gene-related peptide (CGRP) on [3H]-acetylcholine ([3H]-ACh) release from motor nerve endings and its interaction with presynaptic facilitatory A2a-adenosine and nicotinic acetylcholine receptors was studied on rat phrenic nerve-hemidiaphragm preparations loaded with [3H]-choline. 2. CGRP (100-400 nM) increased electrically evoked [3H]-ACh release from phrenic nerve endings in a concentration-dependent manner. 3. The magnitude of CGRP excitation increased with the increase of the stimulation pulse duration from 40 microseconds to 1 ms, keeping the frequency, the amplitude and the train length constants. With 1 ms pulses, the evoked [3H]-ACh release was more intense than with 40 microseconds pulse duration. 4. Both the nicotinic acetylcholine receptor agonist, 1,1-dimethyl-4-phenylpiperazinium, and the A2a adenosine receptor agonist, CGS 21680C, increased evoked [3H]-ACh release, but only CGS 21680C potentiated the facilitatory effect of CGRP. This potentiation was prevented by the A2a adenosine receptor antagonist, PD 115,199. 5. Adenosine deaminase prevented the excitatory effect of CGRP (400 nM) on [3H]-ACh release. This effect was reversed by the non-hydrolysable A2a-adenosine receptor agonist, CGS 21680C. 6. The nicotinic antagonist, tubocurarine, did not significantly change, whereas the A2-adenosine receptor antagonist, PD 115,199, blocked the CGRP facilitation. The A1-adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, potentiated the CGRP excitatory effect. 7. The results suggest that the facilitatory effect of CGRP on evoked [3H]-ACh release from rat phrenic motor nerve endings depends on the presence of endogenous adenosine which tonically activates A2a-adenosine receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8004402

  2. Wound healing effects of noni (Morinda citrifolia L.) leaves: a mechanism involving its PDGF/A2A receptor ligand binding and promotion of wound closure.

    PubMed

    Palu, Afa; Su, Chen; Zhou, Bing-Nan; West, Brett; Jensen, Jarakae

    2010-10-01

    Morinda citrifolia L. (Rubiaceae) commonly known as noni, has been used in Polynesia by traditional healers for the treatment of cuts, bruises and wounds. Our objective was to investigate the wound-healing mechanisms of the noni leaf. The investigations of its wound-healing mechanisms were carried out using fresh noni leaf juice (NLJ), noni leaf ethanol extract (NLEE) and its methanol (MFEE) and hexane (HFEE) fractions on the PDGF and A(2A) receptors in vitro and topically in mice. Fresh noni leaf juice showed significant affinity to PDGF receptors, and displayed 166% binding inhibition of the ligand binding to its receptors, while at the same concentration, it only had 7% inhibition of the ligand binding to the A(2A) receptors. NLEE, HFEE and MFEE showed significant affinity to A(2A) receptors, concentration dependently, with IC(50) values of 34.1, 42.9 and 86.7 μg/mL, respectively. However, MFEE significantly increased wound closure and reduced the half closure time in mice with a CT(50) of 5.4 ± 0.2 days compared with control (p < 0.05). These results suggest that noni leaf significantly accelerated wound healing in mice via its ligand binding to the PDGF and A(2A) receptors as its probable mechanisms of wound-healing and also support its traditional usage for wound-healing in Polynesia.

  3. MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

    PubMed Central

    Volkmann, Robert A.; Fanger, Christopher M.; Anderson, David R.; Sirivolu, Venkata Ramana; Paschetto, Kathy; Gordon, Earl; Virginio, Caterina; Gleyzes, Melanie; Buisson, Bruno; Steidl, Esther; Mierau, Susanna B.; Fagiolini, Michela; Menniti, Frank S.

    2016-01-01

    GluN2A is the most abundant of the GluN2 NMDA receptor subunits in the mammalian CNS. Physiological and genetic evidence implicate GluN2A-containing receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2A-selective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)pyrazine-2-carboxamide) and MPX-007 (5-(((3-fluoro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)methylpyrazine-2-carboxamide). MPX-004 and MPX-007 inhibit GluN2A-containing NMDA receptors expressed in HEK cells with IC50s of 79 nM and 27 nM, respectively. In contrast, at concentrations that completely inhibited GluN2A activity these compounds have no inhibitory effect on GluN2B or GluN2D receptor-mediated responses in similar HEK cell-based assays. Potency and selectivity were confirmed in electrophysiology assays in Xenopus oocytes expressing GluN2A-D receptor subtypes. Maximal concentrations of MPX-004 and MPX-007 inhibited ~30% of the whole-cell current in rat pyramidal neurons in primary culture and MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices. GluN2A-selectivity at native receptors was confirmed by the finding that MPX-004 had no inhibitory effect on NMDA receptor mediated synaptic currents in cortical slices from GRIN2A knock out mice. Thus, MPX-004 and MPX-007 offer highly selective pharmacological tools to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders. PMID:26829109

  4. Extended characterisation of the serotonin 2A (5-HT2A) receptor-selective PET radiotracer 11C-MDL100907 in humans: quantitative analysis, test-retest reproducibility, and vulnerability to endogenous 5-HT tone

    PubMed Central

    Talbot, Peter S.; Slifstein, Mark; Hwang, Dah-Ren; Huang, Yiyun; Scher, Erica; Abi-Dargham, Anissa; Laruelle, Marc

    2011-01-01

    Introduction scanning properties and analytic methodology of the 5-HT2A receptor-selective positron emission tomography (PET) tracer 11C-MDL100907 have been partially characterised in previous reports. We present an extended characterisation in healthy human subjects. Methods 64 11C-MDL100907 PET scans with metabolite-corrected arterial input function were performed in 39 healthy adults (18–55 yr). 12 subjects were scanned twice (duration 150 min) to provide data on plasma analysis, model order estimation, and stability and test-retest characteristics of outcome measures. All other scans were 90 min duration. 3 subjects completed scanning at baseline and following 5-HT2A receptor antagonist medication (risperidone or ciproheptadine) to provide definitive data on the suitability of the cerebellum as reference region. 10 subjects were scanned under reduced 5-HT and control conditions using rapid tryptophan depletion to investigate vulnerability to competition with endogenous 5-HT. 13 subjects were scanned as controls in clinical protocols. Pooled data were used to analyze the relationship between tracer injected mass and receptor occupancy, and age-related decline in 5-HT2A receptors. Results optimum analytic method was a 2-tissue compartment model with arterial input function. However, basis function implementation of SRTM may be suitable for measuring between-group differences non-invasively and warrants further investigation. Scan duration of 90 minutes achieved stable outcome measures in all cortical regions except orbitofrontal which required 120 minutes. Binding potential (BPP and BPND) test-retest variability was very good (7–11%) in neocortical regions other than orbitofrontal, and moderately good (14–20%) in orbitofrontal cortex and medial temporal lobe. Saturation occupancy of 5-HT2A receptors by risperidone validates the use of the cerebellum as a region devoid of specific binding for the purposes of PET. We advocate a mass limit of 4.6 µg to remain

  5. Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells.

    PubMed

    Wu, Pei-Yu; Lin, Yu-Chia; Chang, Chia-Ling; Lu, Hsing-Tsen; Chin, Chia-Hsuan; Hsu, Tsan-Ting; Chu, Dachen; Sun, Synthia H

    2009-06-01

    Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X7 receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined.We characterized the role of P2X7 receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X7 receptors. Functional inhibition of P2X7 receptors by P2X7 receptor selective antagonists, 5'-triphosphate, periodate-oxidized 2',3'-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X7 receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca2+ concentrations ([Ca2+]i) were decreased in either RA- or oATP-differentiated or P2X7receptor knockdown N2a cells. Simply cultured N2a cells in low Ca2+ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X7 receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X7 receptors are involved in neuronal differentiation.We provide additional evidence shown that the ATP release-activated P2X7 receptor is important in maintaining cell survival of N2a neuroblastoma cells.

  6. Fc gamma receptor 3A and 2A polymorphisms do not predict response to rituximab in follicular lymphoma

    PubMed Central

    Kenkre, Vaishalee P.; Hong, Fangxin; Cerhan, James R.; Lewis, Marcia; Sullivan, Leslie; Williams, Michael E.; Gascoyne, Randy D.; Horning, Sandra J.; Kahl, Brad S.

    2015-01-01

    Purpose Pre-clinical studies suggest that single nucleotide polymorphisms (SNPs) in the Fcγ receptor (FCGR) genes influence response to rituximab, but the clinical relevance of this is uncertain. Experimental Design We prospectively obtained specimens for genotyping in the RESORT study, where 408 previously untreated, low tumor burden follicular lymphoma (FL) patients were treated with single agent rituximab. Patients received rituximab in 4 weekly doses and responders were randomized to rituximab re-treatment (RR) upon progression versus maintenance rituximab (MR). SNP genotyping was performed in 321 consenting patients. Results Response rates to initial therapy and response duration were correlated with the FCGR3A SNP at position 158 (rs396991) and the FCGR2A SNP at position 131 (rs1801274). The response rate to initial rituximab was 71%. No FCGR genotypes or grouping of genotypes were predictive of initial response. 289 patients were randomized to RR (n = 143) or to MR (n = 146). With a median follow up of 5.5 years, the 3-yr response duration in the RR arm and the MR arm was 50% and 78%, respectively. Genotyping was available in 235 of 289 randomized patients. In patients receiving RR (n = 115) or MR (n =120), response duration was not associated with any FCGR genotypes or genotype combinations. Conclusions Based on this analysis of treatment-naïve, low tumor burden FL, we conclude that the FCGR3A and FCGR2A SNPs do not confer differential responsiveness to rituximab. PMID:26510856

  7. Effect of adenosine A(2A) receptor antagonists on L-DOPA-induced hydroxyl radical formation in rat striatum.

    PubMed

    Gołembiowska, Krystyna; Dziubina, Anna; Kowalska, Magdalena; Kamińska, Katarzyna

    2009-02-01

    A(2A) adenosine receptor antagonists have been proposed as a new therapy for Parkinson's disease (PD). Since oxidative stress plays an important role in the pathogenesis of PD, we studied the effect of the selective A(2A) adenosine receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) on L: -3,4-dihydroxyphenylalanine (L: -DOPA)-induced hydroxyl radical generation using in vivo microdialysis in the striatum of freely moving rats. L: -DOPA (100 mg/kg; in the presence of benserazide, 50 mg/kg) given acutely or repeatedly for 14 days generated a high level of hydroxyl radicals, measured by HPLC with electrochemical detection, as the product of their reaction with p-hydroxybenzoic acid (PBA). CSC (1 mg/kg) and ZM 241385 (3 mg/kg) decreased haloperidol (0.5 mg/kg)-induced catalepsy, while at low doses of 0.1 and 0.3 mg/kg, respectively, they did not display an effect. CSC (1 and 5 mg/kg) and ZM 241385 (3 and 9 mg/kg) given acutely, or CSC (1 mg/kg) and ZM 241385 (3 mg/kg) given repeatedly, increased the production of hydroxyl radicals in dialysates from rat striatum. Both acute and repeated administration of CSC (0.1 and 1 mg/kg) and ZM 241385 (3 mg/kg) decreased L: -DOPA-induced generation of hydroxyl radicals. However, a high single dose of either CSC (5 mg/kg) and ZM 241385 (9 mg/kg) markedly potentiated the effect of L: -DOPA on hydroxyl radical production. The increase in hydroxyl radical production by acute and chronic injection of CSC and ZM 241385 may be related to the increased release of dopamine (DA) and its metabolism in striatal dialysates. Similarly, increased DA release following a single high dose of CSC or ZM 241385 appears to be responsible for augmentation of L: -DOPA-induced hydroxyl radical formation. Conversely, the inhibition of L: -DOPA-induced production of hydroxyl radical by single and repeated low doses of CSC or repeated low doses of ZM

  8. 5-HT2A Receptor Binding in the Frontal Cortex of Parkinson's Disease Patients and Alpha-Synuclein Overexpressing Mice: A Postmortem Study.

    PubMed

    Rasmussen, Nadja Bredo; Olesen, Mikkel Vestergaard; Brudek, Tomasz; Plenge, Per; Klein, Anders Bue; Westin, Jenny E; Fog, Karina; Wörtwein, Gitta; Aznar, Susana

    2016-01-01

    The 5-HT2A receptor is highly involved in aspects of cognition and executive function and seen to be affected in neurodegenerative diseases like Alzheimer's disease and related to the disease pathology. Even though Parkinson's disease (PD) is primarily a motor disorder, reports of impaired executive function are also steadily being associated with this disease. Not much is known about the pathophysiology behind this. The aim of this study was thereby twofold: (1) to investigate 5-HT2A receptor binding levels in Parkinson's brains and (2) to investigate whether PD associated pathology, alpha-synuclein (AS) overexpression, could be associated with 5-HT2A alterations. Binding density for the 5-HT2A-specific radioligand [(3)H]-MDL 100.907 was measured in membrane suspensions of frontal cortex tissue from PD patients. Protein levels of AS were further measured using western blotting. Results showed higher AS levels accompanied by increased 5-HT2A receptor binding in PD brains. In a separate study, we looked for changes in 5-HT2A receptors in the prefrontal cortex in 52-week-old transgenic mice overexpressing human AS. We performed region-specific 5-HT2A receptor binding measurements followed by gene expression analysis. The transgenic mice showed lower 5-HT2A binding in the frontal association cortex that was not accompanied by changes in gene expression levels. This study is one of the first to look at differences in serotonin receptor levels in PD and in relation to AS overexpression.

  9. Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats

    PubMed Central

    Alencar, Allan K N; Pereira, Sharlene L; Montagnoli, Tadeu L; Maia, Rodolfo C; Kümmerle, Arthur E; Landgraf, Sharon S; Caruso-Neves, Celso; Ferraz, Emanuelle B; Tesch, Roberta; Nascimento, José H M; de Sant'Anna, Carlos M R; Fraga, Carlos A M; Barreiro, Eliezer J; Sudo, Roberto T; Zapata-Sudo, Gisele

    2013-01-01

    Background and Purpose Pulmonary arterial hypertension (PAH) is characterized by enhanced pulmonary vascular resistance, right ventricular hypertrophy and increased right ventricular systolic pressure. Here, we investigated the effects of a N-acylhydrazone derivative, 3,4-dimethoxyphenyl-N-methyl-benzoylhydrazide (LASSBio-1359), on monocrotaline (MCT)-induced pulmonary hypertension in rats. Experimental Approach PAH was induced in male Wistar rats by a single i.p. injection of MCT (60 mg·kg−1) and 2 weeks later, oral LASSBio-1359 (50 mg·kg−1) or vehicle was given once daily for 14 days. Echocardiography was used to measure cardiac function and pulmonary artery dimensions, with histological assay of vascular collagen. Studies of binding to human recombinant adenosine receptors (A1, A2A, A3) and of docking with A2A receptors were also performed. Key Results MCT administration induced changes in vascular and ventricular structure and function, characteristic of PAH. These changes were reversed by treatment with LASSBio-1359. MCT also induced endothelial dysfunction in pulmonary artery, as measured by diminished relaxation of pre-contracted arterial rings, and this dysfunction was reversed by LASSBio-1359. In pulmonary artery rings from normal Wistar rats, LASSBio-1359 induced relaxation, which was decreased by the adenosine A2A receptor antagonist, ZM 241385. In adenosine receptor binding studies, LASSBio-1359 showed most affinity for the A2A receptor and in the docking analyses, binding modes of LASSBio-1359 and the A2A receptor agonist, CGS21680, were very similar. Conclusion and Implications In rats with MCT-induced PAH, structural and functional changes in heart and pulmonary artery were reversed by treatment with oral LASSBio-1359, most probably through the activation of adenosine A2A receptors. PMID:23530610

  10. Differential regulation of 5-HT2A receptor mRNA expression following withdrawal from a chronic escalating dose regimen of D-amphetamine.

    PubMed

    Horner, Kristen A; Gilbert, Yamiece E; Noble, Erika S

    2011-05-16

    Several lines of evidence indicate that psychostimulant withdrawal can induce negative emotional symptoms, such as anhedonia and dysphoria, which may be due in part, to dysfunction of the serotonin (5-HT) system, including alterations in 5-HT receptors. For example, changes in 5-HT(2A) receptor function in prefrontal cortex (PFC) have been reported in association with psychostimulant withdrawal. However, it is not known if alterations in 5-HT(2A) receptor mRNA expression occur in the PFC or other limbic-associated areas following withdrawal from chronic psychostimulant treatment. The goal of the current study was to determine the effects of chronic, escalating doses of D-amphetamine (D-AMPH) and withdrawal on the expression of 5-HT(2A) receptors in the cortex, caudate putamen, NAc and hippocampus of rat brain. Animals were treated three times a day for 4 days with escalating doses of D-AMPH (1-10 mg/kg). Twenty-four hours after the final dose of D-AMPH, animals were sacrificed and the tissue processed for in situ hybridization histochemistry. Chronic, escalating doses of D-AMPH, followed by a 24 h withdrawal period, significantly decreased 5-HT(2A) receptor mRNA expression in the prefrontal, motor and cingulate cortices, while 5-HT(2A) receptor mRNA expression in the NAc, caudal CPu and hippocampus were significantly increased. These data indicate that region-specific changes in 5-HT(2A) receptor mRNA expression occur in limbic system and associated areas following chronic D-AMPH treatment, supporting the notion that alterations in the 5-HT system may contribute to the negative emotional aspects of psychostimulant withdrawal.

  11. Hyperthermia-induced seizures alter adenosine A1 and A2A receptors and 5'-nucleotidase activity in rat cerebral cortex.

    PubMed

    León-Navarro, David Agustín; Albasanz, José L; Martín, Mairena

    2015-08-01

    Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia-induced seizures on adenosine A1 and A2A receptors and 5'-nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real-time PCR, and 5'-nucleotidase activity assays. Hyperthermic seizures were induced in 13-day-old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A1 receptor density was observed using [(3) H]DPCPX as radioligand, and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density was detected, using [(3) H]ZM241385 as radioligand, 48 h after hyperthermia-evoked convulsions. These short-term changes in A1 and A2A receptors were also accompanied by a loss of 5'-nucleotidase activity. No significant variations either in A1 or A2A receptor density or 5'-nucleotidase were observed 5 and 20 days after hyperthermic seizures. Taken together, both regulation of A1 and A2A receptors and loss of 5'-nucleotidase in the cerebral cortex suggest the existence of a neuroprotective mechanism against seizures. Febrile seizure is one of the most common convulsive disorders in children. The consequences of hyperthermia-induced seizures (animal model of febrile seizures) on adenosine A1 and A2A receptors and 5'-nucleotidase activity have been studied at different periods in cerebral cortical area. A significant increase in A1 receptor density and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density and 5'-nucleotidase activity was detected 48 h after convulsions evoked by hyperthermia

  12. RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells.

    PubMed

    Luessen, Deborah J; Hinshaw, Tyler P; Sun, Haiguo; Howlett, Allyn C; Marrs, Glen; McCool, Brian A; Chen, Rong

    2016-11-01

    Dysregulated expression and function of dopamine D2 receptors (D2Rs) are implicated in drug addiction, Parkinson's disease and schizophrenia. In the current study, we examined whether D2Rs are modulated by regulator of G protein signaling 2 (RGS2), a member of the RGS family that regulates G protein signaling via acceleration of GTPase activity. Using neuroblastoma 2a (N2A) cells, we found that RGS2 was immunoprecipitated by aluminum fluoride-activated Gαi2 proteins. RGS2 siRNA knockdown enhanced membrane [(35)S] GTPγS binding to activated Gαi/o proteins, augmented inhibition of cAMP accumulation and increased ERK phosphorylation in the presence of a D2/D3R agonist quinpirole when compared to scrambled siRNA treatment. These data suggest that RGS2 is a negative modulator of D2R-mediated Gαi/o signaling. Moreover, RGS2 knockdown slightly increased constitutive D2R internalization and markedly abolished quinpirole-induced D2R internalization assessed by immunocytochemistry. RGS2 knockdown did not compromise agonist-induced β-arrestin membrane recruitment; however, it prevents β-arrestin dissociation from the membrane after prolonged quinpirole treatment during which time β-arrestin moved away from the membrane in control cells. Additionally, confocal microscopy analysis of β-arrestin post-endocytic fate revealed that quinpirole treatment caused β-arrestin to translocate to the early and the recycling endosome in a time-dependent manner in control cells whereas translocation of β-arrestin to these endosomes did not occur in RGS2 knockdown cells. The impaired β-arrestin translocation likely contributed to the abolishment of quinpirole-stimulated D2R internalization in RGS2 knockdown cells. Thus, RGS2 is integral for β-arrestin-mediated D2R internalization. The current study revealed a novel regulation of D2R signaling and internalization by RGS2 proteins.

  13. Expression of 5-HT2A receptors in prefrontal cortex pyramidal neurons projecting to nucleus accumbens. Potential relevance for atypical antipsychotic action.

    PubMed

    Mocci, Giuseppe; Jiménez-Sánchez, Laura; Adell, Albert; Cortés, Roser; Artigas, Francesc

    2014-04-01

    The prefrontal cortex (PFC) is involved in higher brain functions altered in schizophrenia. Classical antipsychotic drugs modulate information processing in cortico-limbic circuits via dopamine D2 receptor blockade in nucleus accumbens (NAc) whereas atypical antipsychotic drugs preferentially target cortical serotonin (5-HT) receptors. The brain networks involved in the therapeutic action of atypical drugs are not fully understood. Previous work indicated that medial PFC (mPFC) pyramidal neurons projecting to ventral tegmental area express 5-HT2A receptors suggesting that atypical antipsychotic drugs modulate dopaminergic activity distally, via 5-HT2A receptor (5-HT2A-R) blockade in PFC. Since the mPFC also projects heavily to NAc, we examined whether NAc-projecting pyramidal neurons also express 5-HT2A-R. Using a combination of retrograde tracing experiments and in situ hybridization we report that a substantial proportion of mPFC-NAc pyramidal neurons in rat brain express 5-HT2A-R mRNA in a layer- and area-specific manner (up to 68% in layer V of contralateral cingulate). The functional relevance of 5-HT2A-R to modulate mPFC-NAc projections was examined in dual-probe microdialysis experiments. The application of the preferential 5-HT2A-R agonist DOI into mPFC enhanced glutamate release locally (+66 ± 18%) and in NAc (+74 ± 12%) indicating that cortical 5-HT2A-R activation augments glutamatergic transmission in NAc. Since NAc integrates glutamatergic and dopaminergic inputs, blockade of 5-HT2A-R by atypical drugs may reduce cortical excitatory inputs onto GABAergic neurons of NAc, adding to dopamine D2 receptor blockade. Together with previous observations, the present results suggest that atypical antipsychotic drugs may control the activity of the mesolimbic pathway at cell body and terminal level.

  14. 2-Amino-N-pyrimidin-4-ylacetamides as A2A receptor antagonists: 2. Reduction of hERG activity, observed species selectivity, and structure-activity relationships.

    PubMed

    Slee, Deborah H; Moorjani, Manisha; Zhang, Xiaohu; Lin, Emily; Lanier, Marion C; Chen, Yongsheng; Rueter, Jaimie K; Lechner, Sandra M; Markison, Stacy; Malany, Siobhan; Joswig, Tanya; Santos, Mark; Gross, Raymond S; Williams, John P; Castro-Palomino, Julio C; Crespo, María I; Prat, Maria; Gual, Silvia; Díaz, José-Luis; Jalali, Kayvon; Sai, Yang; Zuo, Zhiyang; Yang, Chun; Wen, Jenny; O'Brien, Zhihong; Petroski, Robert; Saunders, John

    2008-03-27

    Previously we have described a series of novel A 2A receptor antagonists with excellent water solubility. As described in the accompanying paper, the antagonists were first optimized to remove an unsubstituted furyl moiety, with the aim of avoiding the potential metabolic liabilities that can arise from the presence of an unsubstituted furan. This effort identified a series of potent and selective methylfuryl derivatives. Herein, we describe the further optimization of this series to increase potency, maintain selectivity for the human A 2A vs the human A 1 receptor, and minimize activity against the hERG channel. In addition, the observed structure-activity relationships against both the human and the rat A 2A receptor are reported.

  15. Allosteric signaling through an mGlu2 and 5-HT2A heteromeric receptor complex and its potential contribution to schizophrenia

    PubMed Central

    Moreno, José L.; Miranda-Azpiazu, Patricia; García-Bea, Aintzane; Younkin, Jason; Cui, Meng; Kozlenkov, Alexey; Ben-Ezra, Ariel; Voloudakis, Georgios; Fakira, Amanda K.; Baki, Lia; Ge, Yongchao; Georgakopoulos, Anastasios; Morón, José A.; Milligan, Graeme; López-Giménez, Juan F.; Robakis, Nikolaos K.; Logothetis, Diomedes E.; Meana, J. Javier; González-Maeso, Javier

    2016-01-01

    Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) can form multiprotein complexes (heteromers), which can alter the pharmacology and functions of the constituent receptors. Previous findings demonstrated that the Gq/11-coupled serotonin 5-HT2A receptor and the Gi/o-coupled metabotropic glutamate 2 (mGlu2) receptor—GPCRs that are involved in signaling alterations associated with psychosis—assemble into a heteromeric complex in the mammalian brain. In single-cell experiments with various mutant versions of the mGlu2 receptor, we showed that stimulation of cells expressing mGlu2–5-HT2A heteromers with an mGlu2 agonist led to activation of Gq/11 proteins by the 5-HT2A receptors. For this crosstalk to occur, one of the mGlu2 subunits had to couple to Gi/o proteins, and we determined the relative location of the Gi/o-contacting subunit within the mGlu2 homodimer of the heteromeric complex. Additionally, mGlu2-dependent activation of Gq/11, but not Gi/o, was reduced in the frontal cortex of 5-HT2A knockout mice and was reduced in the frontal cortex of postmortem brains from schizophrenic patients. These findings offer structural insights into this important target in molecular psychiatry. PMID:26758213

  16. Activation of 5-HT2a receptors in the basolateral amygdala promotes defeat-induced anxiety and the acquisition of conditioned defeat in Syrian hamsters.

    PubMed

    Clinard, Catherine T; Bader, Lauren R; Sullivan, Molly A; Cooper, Matthew A

    2015-03-01

    Conditioned defeat is a model in Syrian hamsters (Mesocricetus auratus) in which normal territorial aggression is replaced by increased submissive and defensive behavior following acute social defeat. The conditioned defeat response involves both a fear-related memory for a specific opponent as well as anxiety-like behavior indicated by avoidance of novel conspecifics. We have previously shown that systemic injection of a 5-HT2a receptor antagonist reduces the acquisition of conditioned defeat. Because neural activity in the basolateral amygdala (BLA) is critical for the acquisition of conditioned defeat and BLA 5-HT2a receptors can modulate anxiety but have a limited effect on emotional memories, we investigated whether 5-HT2a receptor modulation alters defeat-induced anxiety but not defeat-related memories. We injected the 5-HT2a receptor antagonist MDL 11,939 (0 mM, 1.7 mM or 17 mM) or the 5-HT2a receptor agonist TCB-2 (0 mM, 8 mM or 80 mM) into the BLA prior to social defeat. We found that injection of MDL 11,939 into the BLA impaired acquisition of the conditioned defeat response and blocked defeat-induced anxiety in the open field, but did not significantly impair avoidance of former opponents in the Y-maze. Furthermore, we found that injection of TCB-2 into the BLA increased the acquisition of conditioned defeat and increased anxiety-like behavior in the open field, but did not alter avoidance of former opponents. Our data suggest that 5-HT2a receptor signaling in the BLA is both necessary and sufficient for the development of conditioned defeat, likely via modulation of defeat-induced anxiety.

  17. Spatiotemporal brain dynamics of emotional face processing modulations induced by the serotonin 1A/2A receptor agonist psilocybin.

    PubMed

    Bernasconi, Fosco; Schmidt, André; Pokorny, Thomas; Kometer, Michael; Seifritz, Erich; Vollenweider, Franz X

    2014-12-01

    Emotional face processing is critically modulated by the serotonergic system. For instance, emotional face processing is impaired by acute psilocybin administration, a serotonin (5-HT) 1A and 2A receptor agonist. However, the spatiotemporal brain mechanisms underlying these modulations are poorly understood. Here, we investigated the spatiotemporal brain dynamics underlying psilocybin-induced modulations during emotional face processing. Electrical neuroimaging analyses were applied to visual evoked potentials in response to emotional faces, following psilocybin and placebo administration. Our results indicate a first time period of strength (i.e., Global Field Power) modulation over the 168-189 ms poststimulus interval, induced by psilocybin. A second time period of strength modulation was identified over the 211-242 ms poststimulus interval. Source estimations over these 2 time periods further revealed decreased activity in response to both neutral and fearful faces within limbic areas, including amygdala and parahippocampal gyrus, and the right temporal cortex over the 168-189 ms interval, and reduced activity in response to happy faces within limbic and right temporo-occipital brain areas over the 211-242 ms interval. Our results indicate a selective and temporally dissociable effect of psilocybin on the neuronal correlates of emotional face processing, consistent with a modulation of the top-down control.

  18. Neuroprotective potential of adenosine A2A and cannabinoid CB1 receptor antagonists in an animal model of Parkinson disease.

    PubMed

    Cerri, Silvia; Levandis, Giovanna; Ambrosi, Giulia; Montepeloso, Elena; Antoninetti, Gian Filippo; Franco, Rafael; Lanciego, José Luis; Baqi, Younis; Müller, Christa E; Pinna, Annalisa; Blandini, Fabio; Armentero, Marie Therese

    2014-05-01

    The development of nondopaminergic therapeutic strategies that may improve motor and nonmotor deficits, while possibly slowing down the neurodegenerative process and associated neuroinflammation,is a primary goal of Parkinson disease (PD) research. We investigated the neuroprotective and anti-inflammatory potential of combined and single treatment with adenosine A2A and cannabinoid CB1 receptor antagonists MSX-3 and rimonabant, respectively, in a rodent model of PD. Rats bearing a unilateral intrastriatal 6-hydroxydopamine lesion were treated chronically with MSX-3 (0.5or 1 mg/kg/d) and rimonabant (0.1 mg/kg/d) given as monotherapy or combined. The effects of the treatments to counteract dopaminergic cell death and neuroinflammation were assessed by immunohistochemistry for tyrosine hydroxylase and glial cell markers, respectively. Both rimonabant and MSX-3 (1 mg/kg/d) promoted dopaminergic neuron survival in the substantia nigra pars compacta (SNc) when given alone; this effect was weakened when the compounds were combined. Glial activation was not significantly affected by MSX-3 (1 mg/kg/d), whereas rimonabant seemed to increase astrocyte cell density in the SNc. Our findings demonstrate the neuroprotective potential of single treatments and suggest that glial cells might be involved in this protective effect. The results also indicate that the neuroprotective potential of combined therapy may not necessarily reflect or promote single-drug effects and point out that special care should be taken when considering multidrug therapies in PD.

  19. Involvement of adenosine A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of cocaine and methamphetamine in rats.

    PubMed

    Justinova, Zuzana; Ferre, Sergi; Segal, Pavan N; Antoniou, Katerina; Solinas, Marcello; Pappas, Lara A; Highkin, Jena L; Hockemeyer, Jorg; Munzar, Patrik; Goldberg, Steven R

    2003-12-01

    Adenosine, by acting on adenosine A1 and A2A receptors, is known to antagonistically modulate dopaminergic neurotransmission. We have recently reported that nonselective adenosine receptor antagonists (caffeine and 3,7-dimethyl-1-propargylxanthine) can partially substitute for the discriminative-stimulus effects of methamphetamine. In the present study, by using more selective compounds, we investigated the involvement of A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of both cocaine and methamphetamine. The effects of the A1 receptor agonist N6-cyclopentyladenosine (CPA; 0.01-0.1 mg/kg) and antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 1.3-23.7 mg/kg) and the A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680; 0.03-0.18 mg/kg) and antagonist 3-(3-hydroxypropyl)-8-(3-methoxystyryl)-7-methyl-1-propargylxanthin phosphate disodium salt (MSX-3; 1-56 mg/kg) were evaluated in rats trained to discriminate either 1 mg/kg methamphetamine or 10 mg/kg cocaine from saline under a fixed-ratio 10 schedule of food presentation. The A1 and A2A receptor antagonists (CPT and MSX-3) both produced high levels of drug-lever selection when substituted for either methamphetamine or cocaine and significantly shifted dose-response curves of both psychostimulants to the left. Unexpectedly, the A2A receptor agonist CGS 21680 also produced drug-appropriate responding (although at lower levels) when substituted for the cocaine-training stimulus, and both CGS 21680 and the A1 receptor agonist CPA significantly shifted the cocaine dose-response curve to the left. In contrast, both agonists did not produce significant levels of drug-lever selection when substituted for the methamphetamine-training stimulus and failed to shift the methamphetamine dose-response curve. Therefore, adenosine A1 and A2A receptors appear to play important but differential roles in the modulation of the

  20. Differential Expression of Adenosine A1 and A2A Receptors After Upper Cervical (C2) Spinal Cord Hemisection in Adult Rats

    PubMed Central

    Petrov, Theodor; Kreipke, Christian; Alilain, Warren; Nantwi, Kwaku D

    2007-01-01

    Background: In an animal model of spinal cord injury, a latent respiratory motor pathway can be pharmacologically activated via adenosine receptors to restore respiratory function after cervical (C2) spinal cord hemisection that paralyzes the hemidiaphragm ipsilateral to injury. Although spinal phrenic motoneurons immunopositive for adenosine receptors have been demonstrated (C3–C5), it is unclear if adenosine receptor protein levels are altered after C2 hemisection and theophylline administration. Objective: To assess the effects of C2 spinal cord hemisection and theophylline administration on the expression of adenosine receptor proteins. Methods: Adenosine A1 and A2A receptor protein levels were assessed in adult rats classified as (a) noninjured and theophylline treated, (b) C2 hemisected, (c) C2 hemisected and administered theophylline orally (3× daily) for 3 days only, and (d) C2 hemisected and administered theophylline (3× daily for 3 days) and assessed 12 days after drug administration. Assessment of A1 protein levels was carried out via immunohistochemistry and A2A protein levels by densitometry. Results: Adenosine A1 protein levels decreased significantly (both ipsilateral and contralateral to injury) after C2 hemisection; however, the decrease was attenuated in hemisected and theophylline-treated animals. Attenuation in adenosine A1 receptor protein levels persisted when theophylline administration was stopped for 12 days prior to assessment. Adenosine A2A protein levels were unchanged by C2 hemisection; however, theophylline reduced the levels within the phrenic motoneurons. Furthermore, the decrease in A2A levels persisted 12 days after theophylline was withdrawn. Conclusion: Our findings suggest that theophylline mitigates the effects of C2 hemisection by attenuating the C2 hemisection–induced decrease in A1 protein levels. Furthermore, A2A protein levels are unaltered by C2 hemisection but decrease after continuous or interrupted theophylline

  1. MDMA-induced loss of parvalbumin interneurons within the dentate gyrus is mediated by 5HT2A and NMDA receptors.

    PubMed

    Collins, Stuart A; Gudelsky, Gary A; Yamamoto, Bryan K

    2015-08-15

    MDMA is a widely abused psychostimulant which causes a rapid and robust release of the monoaminergic neurotransmitters dopamine and serotonin. Recently, it was shown that MDMA increases extracellular glutamate concentrations in the dorsal hippocampus, which is dependent on serotonin release and 5HT2A/2C receptor activation. The increased extracellular glutamate concentration coincides with a loss of parvalbumin-immunoreactive (PV-IR) interneurons of the dentate gyrus region. Given the known susceptibility of PV interneurons to excitotoxicity, we examined whether MDMA-induced increases in extracellular glutamate in the dentate gyrus are necessary for the loss of PV cells in rats. Extracellular glutamate concentrations increased in the dentate gyrus during systemic and local administration of MDMA. Administration of the NMDA receptor antagonist, MK-801, during systemic injections of MDMA, prevented the loss of PV-IR interneurons seen 10 days after MDMA exposure. Local administration of MDL100907, a selective 5HT2A receptor antagonist, prevented the increases in glutamate caused by reverse dialysis of MDMA directly into the dentate gyrus and prevented the reduction of PV-IR. These findings provide evidence that MDMA causes decreases in PV within the dentate gyrus through a 5HT2A receptor-mediated increase in glutamate and subsequent NMDA receptor activation.

  2. A novel conjugated agent between dopamine and an A2A adenosine receptor antagonist as a potential anti-Parkinson multitarget approach.

    PubMed

    Dalpiaz, Alessandro; Cacciari, Barbara; Vicentini, Chiara Beatrice; Bortolotti, Fabrizio; Spalluto, Giampiero; Federico, Stephanie; Pavan, Barbara; Vincenzi, Fabrizio; Borea, Pier Andrea; Varani, Katia

    2012-03-05

    We propose a potential antiparkinsonian prodrug DP-L-A(2A)ANT (2) obtained by amidic conjugation of dopamine (1) via a succinic spacer to a new triazolo-triazine A(2A) adenosine receptor (AR) antagonist A(2A)ANT (3). The affinity of 2 and its hydrolysis products-1, 3, dopamine-linker DP-L (4) and A(2A)ANT-linker L-A(2A)ANT (5)-was evaluated for hA(1), hA(2A), hA(2B) and hA(3) ARs and rat striatum A(2A)ARs or D(2) receptors. The hydrolysis patterns of 2, 4 and 5 and the stabilities of 1 and 3 were evaluated by HPLC analysis in human whole blood and rat brain homogenates. High hA(2A) affinity was shown by compounds 2 (K(i) = 7.32 ± 0.65 nM), 3 (K(i) = 35 ± 3 nM) and 5 (K(i) = 72 ± 5 nM), whose affinity values were similar in rat striatum. These compounds were not able to change dopamine affinity for D(2) receptors but counteracted the CGS 21680-induced reduction of dopamine affinity. DP-L (4) was inactive on adenosine and dopaminergic receptors. As for stability studies, compounds 4 and 5 were not degraded in incubation media. In human blood, the prodrug 2 was hydrolyzed (half-life = 2.73 ± 0.23 h) mainly on the amidic bound coupling the A(2A)ANT (3), whereas in rat brain homogenates the prodrug 2 was hydrolyzed (half-life > eight hours) exclusively on the amidic bound coupling dopamine, allowing its controlled release and increasing its poor stability as characterized by half-life = 22.5 ± 1.5 min.

  3. Synthesis and pharmacological evaluation of dual acting ligands targeting the adenosine A2A and dopamine D2 receptors for the potential treatment of Parkinson's disease.

    PubMed

    Jörg, Manuela; May, Lauren T; Mak, Frankie S; Lee, Kiew Ching K; Miller, Neil D; Scammells, Peter J; Capuano, Ben

    2015-01-22

    A relatively new strategy in drug discovery is the development of dual acting ligands. These molecules are potentially able to interact at two orthosteric binding sites of a heterodimer simultaneously, possibly resulting in enhanced subtype selectivity, higher affinity, enhanced or modified physiological response, and reduced reliance on multiple drug administration regimens. In this study, we have successfully synthesized a series of classical heterobivalent ligands as well as a series of more integrated and "drug-like" dual acting molecules, incorporating ropinirole as a dopamine D2 receptor agonist and ZM 241385 as an adenosine A2A receptor antagonist. The best compounds of our series maintained the potency of the original pharmacophores at both receptors (adenosine A2A and dopamine D2). In addition, the integrated dual acting ligands also showed promising results in preliminary blood-brain barrier permeability tests, whereas the classical heterobivalent ligands are potentially more suited as pharmacological tools.

  4. Familial risk for mood disorder and the personality risk factor, neuroticism, interact in their association with frontolimbic serotonin 2A receptor binding.

    PubMed

    Frokjaer, Vibe G; Vinberg, Maj; Erritzoe, David; Baaré, William; Holst, Klaus Kähler; Mortensen, Erik Lykke; Arfan, Haroon; Madsen, Jacob; Jernigan, Terry L; Kessing, Lars Vedel; Knudsen, Gitte Moos

    2010-04-01

    Life stress is a robust risk factor for later development of mood disorders, particularly for individuals at familial risk. Likewise, scoring high on the personality trait neuroticism is associated with an increased risk for mood disorders. Neuroticism partly reflects stress vulnerability and is positively correlated to frontolimbic serotonin 2A (5-HT(2A)) receptor binding. Here, we investigate whether neuroticism interacts with familial risk in relation to frontolimbic 5-HT(2A) receptor binding. Twenty-one healthy twins with a co-twin history of mood disorder and 16 healthy twins without a co-twin history of mood disorder were included. They answered self-report personality questionnaires and underwent [(18)F]altanserin positron emission tomography. We found a significant interaction between neuroticism and familial risk in predicting the frontolimbic 5-HT(2A) receptor binding (p=0.026) in an analysis adjusting for age and body mass index. Within the high-risk group only, neuroticism and frontolimbic 5-HT(2A) receptor binding was positively associated (p=0.0037). In conclusion, our data indicate that familial risk and neuroticism interact in their relation to frontolimbic 5-HT(2A) receptor binding. These findings point at a plausible neurobiological link between genetic and personality risk factors and vulnerability to developing mood disorders. It contributes to our understanding of why some people at high risk develop mood disorders while others do not. We speculate that an increased stress reactivity in individuals at high familial risk for mood disorders might enhance the effect of neuroticism in shaping the impact of potential environmental stress and thereby influence serotonergic neurotransmission.

  5. Activation of serotonin2A receptors in the medial septum-diagonal band of Broca complex enhanced working memory in the hemiparkinsonian rats.

    PubMed

    Li, Li-Bo; Zhang, Li; Sun, Yi-Na; Han, Ling-Na; Wu, Zhong-Heng; Zhang, Qiao-Jun; Liu, Jian

    2015-04-01

    Serotonin2A (5-HT2A) receptors are highly expressed in the medial septum-diagonal band of Broca complex (MS-DB), especially in parvalbumin (PV)-positive neurons linked to hippocampal theta rhythm, which is involved in cognition. Cognitive impairments commonly occur in Parkinson's disease. Here we performed behavioral, electrophysiological, neurochemical and immunohistochemical studies in rats with complete unilateral 6-hydroxydopamine lesions of the medial forebrain bundle (MFB) to assess the importance of dopamine (DA) depletion and MS-DB 5-HT2A receptors for working memory. The MFB lesions resulted in working memory impairment and decreases in firing rate and density of MS-DB PV-positive neurons, peak frequency of hippocampal theta rhythm, and DA levels in septohippocampal system and medial prefrontal cortex (mPFC) compared to control rats. Intra-MS-DB injection of high affinity 5-HT2A receptor agonist TCB-2 enhanced working memory, increased firing rate of PV-positive neurons and peak frequency of hippocampal theta rhythm, elevated DA levels in the hippocampus and mPFC, and decreased 5-HT level in the hippocampus in control and lesioned rats. Compared to control rats, the duration of the excitatory effect produced by TCB-2 on the firing rate of PV-positive neurons was markedly shortened in lesioned rats, indicating dysfunction of 5-HT2A receptors. These findings suggest that unilateral lesions of the MFB in rats induced working memory deficit, and activation of MS-DB 5-HT2A receptors enhanced working memory, which may be due to changes in the activity of septohippocampal network and monoamine levels in the hippocampus and mPFC.

  6. Role of serotonin 5-HT2A receptors in the development of cardiac hypertrophy in response to aortic constriction in mice.

    PubMed

    Lairez, O; Cognet, T; Schaak, S; Calise, D; Guilbeau-Frugier, C; Parini, A; Mialet-Perez, J

    2013-06-01

    Serotonin, in addition to its fundamental role as a neurotransmitter, plays a critical role in the cardiovascular system, where it is thought to be involved in the development of cardiac hypertrophy and failure. Indeed, we recently found that mice with deletion of monoamine oxidase A had enhanced levels of blood and cardiac 5-HT, which contributed to exacerbation of hypertrophy in a model of experimental pressure overload. 5-HT2A receptors are expressed in the heart and mediate a hypertrophic response to 5-HT in cardiac cells. However, their role in cardiac remodeling in vivo and the signaling pathways associated are not well understood. In the present study, we evaluated the effect of a selective 5-HT2A receptor antagonist, M100907, on the development of cardiac hypertrophy induced by transverse aortic constriction (TAC). Cardiac 5-HT2A receptor expression was transiently increased after TAC, and was recapitulated in cardiomyocytes, as observed with 5-HT2A in situ labeling by immunohistochemistry. Selective blockade of 5-HT2A receptors prevented the development of cardiac hypertrophy, as measured by echocardiography, cardiomyocyte area and heart weight-to-body weight ratio. Interestingly, activation of calmodulin kinase (CamKII), which is a core mechanism in cardiac hypertrophy, was reduced in cardiac samples from M100907-treated TAC mice compared to vehicle-treated mice. In addition, phosphorylation of histone deacetylase 4 (HDAC4), a downstream partner of CamKII was significantly diminished in M100907-treated TAC mice. Thus, our results show that selective blockade of 5-HT2A receptors has beneficial effect in the development of cardiac hypertrophy through inhibition of the CamKII/HDAC4 pathway.

  7. Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

    SciTech Connect

    Naranjo, Andrea N.; McNeely, Patrick M.; Katsaras, John; Skaja Robinson, Anne

    2016-05-27

    The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, or DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. Furthermore, the studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.

  8. Adenosine A2A receptor antagonism and genetic deletion attenuate the effects of dopamine D2 antagonism on effort-based decision making in mice.

    PubMed

    Pardo, M; Lopez-Cruz, L; Valverde, O; Ledent, C; Baqi, Y; Müller, C E; Salamone, J D; Correa, M

    2012-04-01

    Brain dopamine (DA) and adenosine interact in the regulation of behavioral activation and effort-related processes. In the present studies, a T-maze task was developed in mice for the assessment of effort-related decision making. With this task, the two arms of the maze have different reinforcement densities, and a vertical barrier is positioned in the arm with the higher density (HD), presenting the animal with an effort-related challenge. Under control conditions mice prefer the HD arm, and climb the barrier to obtain the larger amount of food. The DA D(2) receptor antagonist haloperidol decreased selection of the HD arm and increased selection of the arm with the low density of reinforcement. However, the HD arm was still the preferred choice in haloperidol-treated mice trained with barriers in both arms. Pre-feeding the mice to reduce food motivation dramatically increased omissions, an effect that was distinct from the actions of haloperidol. Co-administration of theophylline, a nonselective adenosine receptor antagonist, partially reversed the effects of haloperidol. This effect seems to be mediated by the A(2A) receptor but not the A(1) receptor, since the A(2A) antagonist MSX-3, but not the A(1) antagonist CPT, dose dependently reversed the effects of haloperidol on effort-related choice and on c-Fos expression in the dorsal striatum and nucleus accumbens. In addition, adenosine A(2A) receptor knockout mice were resistant to the effects of haloperidol on effort-related choice in the maze. These results indicate that DA D(2) and adenosine A(2A) receptors interact to regulate effort-related decision making and effort expenditure in mice.

  9. Involvement of serotonin 2A receptor activation in modulating medial prefrontal cortex and amygdala neuronal activation during novelty-exposure.

    PubMed

    Hervig, Mona El-Sayed; Jensen, Nadja Cecilie Hvid; Rasmussen, Nadja Bredo; Rydbirk, Rasmus; Olesen, Mikkel Vestergaard; Hay-Schmidt, Anders; Pakkenberg, Bente; Aznar, Susana

    2017-03-02

    The medial prefrontal cortex (PFC) plays a major role in executive function by exerting a top-down control onto subcortical areas. Novelty-induced frontal cortex activation is 5-HT2A receptor (5-HT2AR) dependent. Here, we further investigated how blockade of 5-HT2ARs in mice exposed to a novel open-field arena affects medial PFC activation and basolateral amygdala (BLA) reactivity. We used c-Fos immunoreactivity (IR) as a marker of neuronal activation and stereological quantification for obtaining the total number of c-Fos-IR neurons as a measure of regional activation. We further examined the impact of 5-HT2AR blockade on the striatal-projecting BLA neurons. Systemic administration of ketanserin (0.5mg/kg) prior to novel open-field exposure resulted in reduced total numbers of c-Fos-IR cells in dorsomedial PFC areas and the BLA. Moreover, there was a positive correlation between the relative time spent in the centre of the open-field and BLA c-Fos-IR in the ketanserin-treated animals. Unilateral medial PFC lesions blocked this effect, ascertaining an involvement of this frontal cortex area. On the other hand, medial PFC lesioning exacerbated the more anxiogenic-like behaviour of the ketanserin-treated animals, upholding its involvement in modulating averseness. Ketanserin did not affect the number of activated striatal-projecting BLA neurons (measured by number of Cholera Toxin b (CTb) retrograde labelled neurons also being c-Fos-IR) following CTb injection in the ventral striatum. These results support a role of 5-HT2AR activation in modulating mPFC and BLA activation during exposure to a novel environment, which may be interrelated. Conversely, 5-HT2AR blockade does not seem to affect the amygdala-striatal projection.

  10. Adenosine A2A receptor agonist prevents cardiac remodeling and dysfunction in spontaneously hypertensive male rats after myocardial infarction

    PubMed Central

    da Silva, Jaqueline S; Gabriel-Costa, Daniele; Sudo, Roberto T; Wang, Hao; Groban, Leanne; Ferraz, Emanuele B; Nascimento, José Hamilton M; Fraga, Carlos Alberto M; Barreiro, Eliezer J; Zapata-Sudo, Gisele

    2017-01-01

    Background This work evaluated the hypothesis that 3,4-methylenedioxybenzoyl-2-thienylhydrazone (LASSBio-294), an agonist of adenosine A2A receptor, could be beneficial for preventing cardiac dysfunction due to hypertension associated with myocardial infarction (MI). Methods Male spontaneously hypertensive rats (SHR) were randomly divided into four groups (six animals per group): sham-operation (SHR-Sham), and myocardial infarction rats (SHR-MI) were treated orally either with vehicle or LASSBio-294 (10 and 20 mg.kg−1.d−1) for 4 weeks. Echocardiography and in vivo hemodynamic parameters measured left ventricle (LV) structure and function. Exercise tolerance was evaluated using a treadmill test. Cardiac remodeling was accessed by LV collagen deposition and tumor necrosis factor α expression. Results Early mitral inflow velocity was significantly reduced in the SHR-MI group, and there was significant recovery in a dose-dependent manner after treatment with LASSBio-294. Exercise intolerance observed in the SHR-MI group was prevented by 10 mg.kg−1.d−1 of LASS-Bio-294, and exercise tolerance exceeded that of the SHR-Sham group at 20 mg.kg−1.d−1. LV end-diastolic pressure increased after MI, and this was prevented by 10 and 20 mg.kg−1.d−1 of LASSBio-294. Sarcoplasmic reticulum Ca2+ ATPase levels were restored in a dose-dependent manner after treatment with LASSBio-294. Fibrosis and inflammatory processes were also counteracted by LASSBio-294, with reductions in LV collagen deposition and tumor necrosis factor α expression. Conclusion In summary, oral administration of LASSBio-294 after MI in a dose-dependent manner prevented the development of cardiac dysfunction, demonstrating this compound’s potential as an alternative treatment for heart failure in the setting of ischemic heart disease with superimposed chronic hypertension. PMID:28293100

  11. INCREASED 5-HT2A RECEPTOR AVAILABILITY IN THE ORBITOFRONTAL CORTEX OF PHYSICALLY AGGRESSIVE PERSONALITY DISORDERED PATIENTS

    PubMed Central

    Rosell, Daniel R.; Thompson, Judy L.; Slifstein, Mark; Xu, Xiaoyan; Frankle, W. Gordon; New, Antonia S.; Goodman, Marianne; Weinstein, Shauna R.; Laruelle, Marc; Dargham, Anissa Abi; Siever, Larry J.

    2011-01-01

    Background Impulsive physical aggression is a common and problematic feature of many personality disorders. The serotonergic system is known to be involved in the pathophysiology of aggression, and multiple lines of evidence have implicated the 5-HT2A receptor (5-HT2AR). We sought to examine the role of the 5-HT2AR in impulsive aggression specifically in the orbitofrontal cortex (OFC), given that our own studies and an extensive literature indicate that serotonergic disturbances in the OFC are linked to aggression. We have previously hypothesized that increased 5-HT2AR function in the OFC is a state phenomenon which promotes impulsive aggression. Methods 5-HT2AR availability was measured with positron emission tomography and the selective 5-HT2AR antagonist radioligand [11C]MDL100907 in two groups of impulsively aggressive personality disordered patients --14 with current physical aggression, and 15 without current physical aggression --and 25 healthy controls. Clinical ratings of various symptom dimensions were also obtained. Results Orbitofrontal 5-HT2AR availability was greater in patients with current physical aggression compared to patients without current physical aggression and healthy controls; no differences in OFC 5-HT2AR availability were observed between patients without current physical aggression and healthy controls. No significant differences in 5-HT2AR availability were observed in other brain regions examined. Among both groups of impulsively aggressive personality disordered patients combined, OFC 5-HT2AR availability was correlated, specifically, with a state measure of impulsive aggression. Conclusions These findings are consistent with our previously described model in which impulsive aggression is related to dynamic changes in 5-HT2AR function in the OFC. PMID:20434136

  12. Hyperactivity in Childhood as a Predictor of School Performance in Elementary School: Modifying Effect of a Serotonin Receptor Gene (5-HTR2A)

    ERIC Educational Resources Information Center

    Pulkki-Raback, Laura; Pullmann, Helle; Hintsanen, Mirka; Alatupa, Saija; Ravaja, Niklas; Lehtimaki, Terho; Keltikangas-jarvinen, Liisa

    2010-01-01

    Introduction: Genes have been suggested to interact with predictors of school performance, but evidence is scarce. The purpose was to examine whether a hyperactive temperament leads to different school performance, depending on variability in a serotonin receptor gene (5-HTR2A). Method: The participants were a population-based sample of 909 girls…

  13. Effect of fluvoxamine on platelet 5-HT2A receptors as studied by [3H]lysergic acid diethylamide ([3H]LSD) binding in healthy volunteers.

    PubMed

    Spigset, O; Mjörndal, T

    1997-09-01

    Alterations in platelet 5-HT2A receptor characteristics have been reported in major depression as well as in other psychiatric diseases, and some effort has been made to utilize platelet 5-HT2A receptor status as a biological correlate to antidepressant drug response. In order to investigate whether treatment with a selective serotonin reuptake inhibitor affects platelet 5-HT2A receptors, we have studied platelet [3H]lysergic acid diethylamide ([3H]LSD) binding in healthy subjects treated with fluvoxamine in increasing dosage once weekly for 4 weeks. After 1 week of fluvoxamine treatment (25 mg/day), both Bmax and Kd were significantly lower than before the start of the treatment (19.9 versus 25.5 fmol/mg protein, P = 0.005 for Bmax; 0.45 versus 0.93 nM, P = 0.006 for Kd). Bmax returned to baseline during week 2, whereas Kd was lower than the baseline value throughout the treatment period. After discontinuation of fluvoxamine treatment, there was a significant increase in Kd (0.50 nM before discontinuation vs. 1.14 nM after discontinuation; P = 0.001), but not in Bmax. The study demonstrates that fluvoxamine affects platelet 5-HT2A receptor status irrespective of underlying psychiatric disease, and that this effect is evident already after 1 week at a subtherapeutic fluvoxamine dose.

  14. Use of LC/MS to assess brain tracer distribution in preclinical, in vivo receptor occupancy studies: dopamine D2, serotonin 2A and NK-1 receptors as examples.

    PubMed

    Chernet, Eyassu; Martin, Laura J; Li, Dominic; Need, Anne B; Barth, Vanessa N; Rash, Karen S; Phebus, Lee A

    2005-12-12

    High performance liquid chromatography combined with either single quad or triple quad mass spectral detectors (LC/MS) was used to measure the brain distribution of receptor occupancy tracers targeting dopamine D2, serotonin 5-HT2A and neurokinin NK-1 receptors using the ligands raclopride, MDL-100907 and GR205171, respectively. All three non-radiolabeled tracer molecules were easily detectable in discrete rat brain areas after intravenous doses of 3, 3 and 30 microg/kg, respectively. These levels showed a differential brain distribution caused by differences in receptor density, as demonstrated by the observation that pretreatment with compounds that occupy these receptors reduced this differential distribution in a dose-dependent manner. Intravenous, subcutaneous and oral dose-occupancy curves were generated for haloperidol at the dopamine D2 receptor as were oral curves for the antipsychotic drugs olanzapine and clozapine. In vivo dose-occupancy curves were also generated for orally administered clozapine, olanzapine and haloperidol at the cortical 5-HT2A binding site. In vivo occupancy at the striatal neurokinin NK-1 binding site by various doses of orally administered MK-869 was also measured. Our results demonstrate the utility of LC/MS to quantify tracer distribution in preclinical brain receptor occupancy studies.

  15. Caffeine increases striatal dopamine D2/D3 receptor availability in the human brain

    DOE PAGES

    Volkow, N. D.; Wang, G. -J.; Logan, J.; ...

    2015-04-14

    Caffeine, the most widely consumed psychoactive substance in the world, is used to promote wakefulness and enhance alertness. Like other wake-promoting drugs (stimulants and modafinil), caffeine enhances dopamine (DA) signaling in the brain, which it does predominantly by antagonizing adenosine A2A receptors (A2AR). However, it is unclear if caffeine, at the doses consumed by humans, increases DA release or whether it modulates the functions of postsynaptic DA receptors through its interaction with adenosine receptors, which modulate them. We used positron emission tomography and [11C]raclopride (DA D2/D3 receptor radioligand sensitive to endogenous DA) to assess if caffeine increased DA release inmore » striatum in 20 healthy controls. Caffeine (300mg p.o.) significantly increased the availability of D2/D3 receptors in putamen and ventral striatum, but not in caudate, when compared with placebo. In addition, caffeine-induced increases in D2/D3 receptor availability in the ventral striatum were associated with caffeine-induced increases in alertness. Our findings indicate that in the human brain, caffeine, at doses typically consumed, increases the availability of DA D2/D3 receptors, which indicates that caffeine does not increase DA in the striatum for this would have decreased D2/D3 receptor availability. Instead, we interpret our findings to reflect an increase in D2/D3 receptor levels in striatum with caffeine (or changes in affinity). Furthermore, the association between increases in D2/D3 receptor availability in ventral striatum and alertness suggests that caffeine might enhance arousal, in part, by upregulating D2/D3 receptors.« less

  16. Caffeine increases striatal dopamine D2/D3 receptor availability in the human brain.

    PubMed

    Volkow, N D; Wang, G-J; Logan, J; Alexoff, D; Fowler, J S; Thanos, P K; Wong, C; Casado, V; Ferre, S; Tomasi, D

    2015-04-14

    Caffeine, the most widely consumed psychoactive substance in the world, is used to promote wakefulness and enhance alertness. Like other wake-promoting drugs (stimulants and modafinil), caffeine enhances dopamine (DA) signaling in the brain, which it does predominantly by antagonizing adenosine A2A receptors (A2AR). However, it is unclear if caffeine, at the doses consumed by humans, increases DA release or whether it modulates the functions of postsynaptic DA receptors through its interaction with adenosine receptors, which modulate them. We used positron emission tomography and [(11)C]raclopride (DA D2/D3 receptor radioligand sensitive to endogenous DA) to assess if caffeine increased DA release in striatum in 20 healthy controls. Caffeine (300 mg p.o.) significantly increased the availability of D2/D3 receptors in putamen and ventral striatum, but not in caudate, when compared with placebo. In addition, caffeine-induced increases in D2/D3 receptor availability in the ventral striatum were associated with caffeine-induced increases in alertness. Our findings indicate that in the human brain, caffeine, at doses typically consumed, increases the availability of DA D2/D3 receptors, which indicates that caffeine does not increase DA in the striatum for this would have decreased D2/D3 receptor availability. Instead, we interpret our findings to reflect an increase in D2/D3 receptor levels in striatum with caffeine (or changes in affinity). The association between increases in D2/D3 receptor availability in ventral striatum and alertness suggests that caffeine might enhance arousal, in part, by upregulating D2/D3 receptors.

  17. Adenosine A2A receptor antagonists improve deficits in initiation of movement and sensory motor integration in the unilateral 6-hydroxydopamine rat model of Parkinson's disease.

    PubMed

    Pinna, Annalisa; Pontis, Silvia; Borsini, Franco; Morelli, Micaela

    2007-08-01

    Evidence obtained in rodent and primate models of Parkinson's disease (PD) and preliminary clinical trials, indicates that adenosine A(2A) receptor antagonists might represent a promising nondopaminergic therapeutic tool for the treatment of PD. Those studies demonstrated the ability of adenosine A(2A) receptor antagonists to potentiate l-dopa-mediated motor improvement, whereas very little is known about counteraction of specific motor deficits and on the effects of these compounds when administered alone. To this aim we evaluated the effects of different adenosine A(2A) receptor antagonists on initiation of movement deficits, gait impairment and sensory-motor deficits, induced in rats by a unilateral 6-hydroxydopamine lesion of dopaminergic nigrostriatal neurons. The following tests were used: (1) initiation time of stepping; (2) adjusting step (stepping with forelimb was measured as the forelimb was dragged laterally); (3) vibrissae-elicited forelimb placing (as index of sensory-motor integration deficits). Acute administration of the A(2A) receptor antagonists SCH 58261 (5 mg/kg i.p.) and ST 1535 (20 mg/kg i.p.) similarly to l-dopa (6 mg/kg i.p.) counteracted the impairments in the initiation time of stepping test, in the adjusting step and in the vibrissae-elicited forelimb placing induced by the lesion. The intensity of the effect was l-dopa > SCH 58261 > ST 1535. The results provide the first evidence that blockade of A(2A) receptors is effective in antagonizing specific motor deficit induced by DA neuron degeneration, such as initiation of movement and sensory-motor integration deficits, even without l-dopa combined administration.

  18. Enhanced recovery of breathing capacity from combined adenosine 2A receptor inhibition and daily acute intermittent hypoxia after chronic cervical spinal injury

    PubMed Central

    Navarrete-Opazo, A.; Dougherty, B.J.; Mitchell, G.S.

    2016-01-01

    Daily acute intermittent hypoxia (dAIH) improves breathing capacity after C2 spinal hemisection (C2HS) in rats. Since C2HS disrupts spinal serotonergic innervation below the injury, adenosine-dependent mechanisms underlie dAIH-induced functional recovery 2 weeks post-injury. We hypothesized that dAIH-induced functional recovery converts from an adenosine-dependent to a serotonin-dependent, adenosine-constrained mechanism with chronic injury. Eight weeks post-C2HS, rats began dAIH (10, 5-min episodes, 10.5% O2; 5-min intervals; 7 days) followed by AIH 3× per week (3×wAIH) for 8 additional weeks with/without systemic A2A receptor inhibition (KW6002) on each AIH exposure day. Tidal volume (VT) and bilateral diaphragm (Dia) and T2 external intercostal motor activity were assessed in unanesthetized rats breathing air and during maximum chemoreflex stimulation (MCS: 7% CO2, 10.5% O2). Nine weeks post-C2HS, dAIH increased VT versus time controls (p < 0.05), an effect enhanced by KW6002 (p < 0.05). dAIH increased bilateral Dia activity (p < 0.05), and KW6002 enhanced this effect in contralateral (p < 0.05) and ipsilateral Dia activity (p < 0.001), but not T2 inspiratory activity. Functional benefits of combined AIH plus systemic A2A receptor inhibition were maintained for 4 weeks. Thus, in rats with chronic injuries: 1) dAIH improves VT and bilateral diaphragm activity; 2) VT recovery is enhanced by A2A receptor inhibition; and 3) functional recovery with A2A receptor inhibition and AIH “reminders” last 4 weeks. Combined dAIH and A2A receptor inhibition may be a simple, safe, and effective strategy to accelerate/enhance functional recovery of breathing capacity in patients with respiratory impairment from chronic spinal injury. PMID:27079999

  19. Differential effects of presynaptic versus postsynaptic adenosine A2A receptor blockade on Δ9-tetrahydrocannabinol (THC) self-administration in squirrel monkeys.

    PubMed

    Justinová, Zuzana; Redhi, Godfrey H; Goldberg, Steven R; Ferré, Sergi

    2014-05-07

    Different doses of an adenosine A2A receptor antagonist MSX-3 [3,7-dihydro-8-[(1E)-2-(3-ethoxyphenyl)ethenyl]-7 methyl-3-[3-(phosphooxy)propyl-1-(2 propynil)-1H-purine-2,6-dione] were found previously to either decrease or increase self-administration of cannabinoids delta-9-tetrahydrocannabinol (THC) or anandamide in squirrel monkeys. It was hypothesized that the decrease observed with a relatively low dose of MSX-3 was related to blockade of striatal presynaptic A2A receptors that modulate glutamatergic neurotransmission, whereas the increase observed with a higher dose was related to blockade of postsynaptic A2A receptors localized in striatopallidal neurons. This hypothesis was confirmed in the present study by testing the effects of the preferential presynaptic and postsynaptic A2A receptor antagonists SCH-442416 [2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] and KW-6002 [(E)-1, 3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione], respectively, in squirrel monkeys trained to intravenously self-administer THC. SCH-442416 produced a significant shift to the right of the THC self-administration dose-response curves, consistent with antagonism of the reinforcing effects of THC. Conversely, KW-6002 produced a significant shift to the left, consistent with potentiation of the reinforcing effects of THC. These results show that selectively blocking presynaptic A2A receptors could provide a new pharmacological approach to the treatment of marijuana dependence and underscore corticostriatal glutamatergic neurotransmission as a possible main mechanism involved in the rewarding effects of THC.

  20. The role of peripheral 5HT2A and 5HT1A receptors on the orofacial formalin test in rats with persistent temporomandibular joint inflammation.

    PubMed

    Okamoto, K; Imbe, H; Tashiro, A; Kimura, A; Donishi, T; Tamai, Y; Senba, E

    2005-01-01

    The role of peripheral serotonin (5HT) 2A and 5HT1A receptors on the orofacial nocifensive behavioral activities evoked by the injection of formalin into the masseter muscle was evaluated in the rats with persistent temporomandibular joint (TMJ) inflammation evoked by Complete Freund's Adjuvant (CFA). The orofacial nocifensive behavioral activities evoked by the injection of formalin into masseter muscle were significantly enhanced at 1 day (CFA day 1 group) or 7 days (CFA day 7 group) during TMJ inflammation. Pretreatment with local administration of 5HT2A receptor antagonist, ketanserin (0.01, 0.1 mg/rat) into the masseter muscle or systemic administration of ketanserin via i.p. injection (1 mg/kg) reduced the orofacial nocifensive behavioral activities of the late phase evoked by formalin injection into masseter muscle on the side of TMJ inflammation (CFA day 7 group). However, local (0.001-0.1 mg/rat) or systemic (1 mg/kg) administration of 5HT1A receptor antagonist, propranolol, into masseter muscle did not produce the antinociceptive effect in CFA day 7 group. Moreover, local administration of ketanserin (0.1 mg) or propranolol (0.1 mg) into masseter muscle did not inhibit nocifensive orofacial behavior in rats without TMJ inflammation. These data suggest that persistent TMJ inflammation causes the elevation of the orofacial nocifensive behavior, and peripheral 5HT2A receptors play an important role in mediating the deep craniofacial tissue nociception in rats with TMJ inflammation.

  1. Creatine, similarly to ketamine, affords antidepressant-like effects in the tail suspension test via adenosine A₁ and A2A receptor activation.

    PubMed

    Cunha, Mauricio P; Pazini, Francis L; Rosa, Julia M; Ramos-Hryb, Ana B; Oliveira, Ágatha; Kaster, Manuella P; Rodrigues, Ana Lúcia S

    2015-06-01

    The benefits of creatine supplementation have been reported in a broad range of central nervous systems diseases, including depression. A previous study from our group demonstrated that creatine produces an antidepressant-like effect in the tail suspension test (TST), a predictive model of antidepressant activity. Since depression is associated with a dysfunction of the adenosinergic system, we investigated the involvement of adenosine A1 and A2A receptors in the antidepressant-like effect of creatine in the TST. The anti-immobility effect of creatine (1 mg/kg, po) or ketamine (a fast-acting antidepressant, 1 mg/kg, ip) in the TST was prevented by pretreatment of mice with caffeine (3 mg/kg, ip, nonselective adenosine receptor antagonist), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (2 mg/kg, ip, selective adenosine A1 receptor antagonist), and 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo-{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)-phenol (ZM241385) (1 mg/kg, ip, selective adenosine A2A receptor antagonist). In addition, the combined administration of subeffective doses of creatine and adenosine (0.1 mg/kg, ip, nonselective adenosine receptor agonist) or inosine (0.1 mg/kg, ip, nucleoside formed by the breakdown of adenosine) reduced immobility time in the TST. Moreover, the administration of subeffective doses of creatine or ketamine combined with N-6-cyclohexyladenosine (CHA) (0.05 mg/kg, ip, selective adenosine A1 receptor agonist), N-6-[2-(3,5-dimethoxyphenyl)-2-(methylphenyl)ethyl]adenosine (DPMA) (0.1 mg/kg, ip, selective adenosine A2A receptor agonist), or dipyridamole (0.1 μg/mouse, icv, adenosine transporter inhibitor) produced a synergistic antidepressant-like effect in the TST. These results indicate that creatine, similarly to ketamine, exhibits antidepressant-like effect in the TST probably mediated by the activation of both adenosine A1 and A2A receptors, further reinforcing the potential of targeting the purinergic system to the management of mood disorders.

  2. Extensive Rigid Analogue Design Maps the Binding Conformation of Potent N-Benzylphenethylamine 5-HT2A Serotonin Receptor Agonist Ligands

    PubMed Central

    2012-01-01

    Based on the structure of the superpotent 5-HT2A agonist 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine, which consists of a ring-substituted phenethylamine skeleton modified with an N-benzyl group, we designed and synthesized a small library of constrained analogues to identify the optimal arrangement of the pharmacophoric elements of the ligand. Structures consisted of diversely substituted tetrahydroisoquinolines, piperidines, and one benzazepine. Based on the structure of (S,S)-9b, which showed the highest affinity of the series, we propose an optimal binding conformation. (S,S)-9b also displayed 124-fold selectivity for the 5-HT2A over the 5-HT2C receptor, making it the most selective 5-HT2A receptor agonist ligand currently known. PMID:23336049

  3. Role of adenosine A(2A) receptors in modulating synaptic functions and brain levels of BDNF: a possible key mechanism in the pathophysiology of Huntington's disease.

    PubMed

    Tebano, Maria Teresa; Martire, Alberto; Chiodi, Valentina; Ferrante, Antonella; Popoli, Patrizia

    2010-09-01

    In the last few years, accumulating evidence has shown the existence of an important cross-talk between adenosine A(2A) receptors (A(2A)Rs) and brain-derived neurotrophic factor (BDNF). Not only are A(2A)Rs involved in the mechanism of transactivation of BDNF receptor TrkB, they also modulate the effect of BDNF on synaptic transmission, playing a facilitatory and permissive role. The cAMP-PKA pathway, the main transduction system operated by A(2A)Rs, is involved in such effects. Furthermore, a basal tonus of A(2A)Rs is required to allow the regulation of BDNF physiological levels in the brain, as demonstrated by the reduced protein levels measured in A(2A)Rs KO mice. The crucial role of adenosine A(2A)Rs in the maintenance of synaptic functions and BDNF levels will be reviewed here and discussed in the light of possible implications for Huntington's disease therapy, in which a joint impairment of BDNF and A(2A)Rs seems to play a pathogenetic role.

  4. Selective blockade of 5-HT2A receptors attenuates the increased temperature response in brown adipose tissue to restraint stress in rats.

    PubMed

    Ootsuka, Youichirou; Blessing, William W; Nalivaiko, Eugene

    2008-03-01

    Previous studies have demonstrated that 5-HT2A receptors may be involved in the central control of thermoregulation and of the cardiovascular system. Our aim was to test whether these receptors mediate thermogenic and tachycardiac responses induced by acute psychological stress. Three groups of adult male Hooded Wistar rats were instrumented with: (i) a thermistor in the interscapular area (for recording brown adipose tissue temperature) and an ultrasound Doppler probe (to record tail blood flow); (ii) temperature dataloggers to record core body temperature; (iii) ECG electrodes. On the day of the experiment, rats were subjected to a 30-min restraint stress preceded by s.c. injection of either vehicle or SR-46349B (a serotonin 2A receptor antagonist) at doses of 0.01, 0.1 and 1.0 mg/kg. The restraint stress caused a rise in brown adipose tissue temperature (from, mean +/- s.e.m., 36.6 +/- 0.2 to 38.0 +/- 0.2 degrees C), transient cutaneous vasoconstriction (tail blood flow decreased from 12 +/- 2 to 5 +/- 1 cm/s), increase in heart rate (from 303 +/- 15 to 453 +/- 15 bpm at the peak, then reduced to 393 +/- 12 bpm at the steady state), and defaecation (6 +/- 1 pellets per restraint session). The core body temperature was not affected by the restraint. Blockade of 5-HT2A receptors attenuated the increase in brown adipose tissue temperature and transient cutaneous vasoconstriction, but not tachycardia and defaecation elicited by restraint stress. These results indicate that psychological stress causes activation of 5-HT2A receptors in neural pathways that control thermogenesis in the brown adipose tissue and facilitate cutaneous vasoconstriction.

  5. Stimulation of serotonin 2A receptors facilitates consolidation and extinction of fear memory in C57BL/6J mice.

    PubMed

    Zhang, Gongliang; Ásgeirsdóttir, Herborg N; Cohen, Sarah J; Munchow, Alcira H; Barrera, Mercy P; Stackman, Robert W

    2013-01-01

    Excessive fear is a hallmark of several emotional and mental disorders such as phobias and panic disorders. Considerable attention is focused on defining the neurobiological mechanisms of the extinction of conditioned fear memory in an effort to identify mechanisms that may hold clinical significance for remediating aberrant fear memory. Serotonin modulates the acquisition and retention of conditioned emotional memory, and the serotonin 2A receptor (5HT2AR) may be one of the postsynaptic targets mediating such effects. Here we tested the hypothesis that the 5HT2AR regulates the consolidation and extinction of fear memory in male C57BL/6J mice. The influence of 5HT2ARs on memory consolidation was further confirmed with a novel object recognition task. With a trace fear conditioning paradigm, administration of the 5HT2AR agonist TCB-2 (1.0 mg/kg, i.p.) before the extinction test facilitated the acquisition of extinction of fear memory as compared to vehicle treatment. In contrast, administration of the 5HT2AR antagonist MDL 11,939 (0.5 mg/kg, i.p.) delayed the acquisition of extinction of fear memory. Further, the post-conditioning administration of TCB-2 enhanced contextual and cued fear memory, possibly by facilitating the consolidation of fear memory. Administration of TCB-2 also facilitated the acquisition of extinction of fear memory in delay fear conditioned mice. Stimulation or blockade of 5HT2ARs did not affect the encoding or retrieval of conditioned fear memory. Finally, administration of TCB-2 right after training in an object recognition task enhanced the consolidation of object memory. These results suggest that stimulation of 5HT2ARs facilitates the consolidation and extinction of trace and delay cued fear memory and the consolidation of object memory. Blocking the 5HT2AR impairs the acquisition of fear memory extinction. The results support the view that serotonergic activation of the 5HT2AR provides an important modulatory influence on circuits

  6. Liver X receptor alpha mediated genistein induction of human dehydroepiandrosterone sulfotransferase (hSULT2A1) in Hep G2 cells

    SciTech Connect

    Chen, Yue; Zhang, Shunfen; Zhou, Tianyan; Huang, Chaoqun; McLaughlin, Alicia; Chen, Guangping

    2013-04-15

    Cytosolic sulfotransferases are one of the major families of phase II drug metabolizing enzymes. Sulfotransferase-catalyzed sulfonation regulates hormone activities, metabolizes drugs, detoxifies xenobiotics, and bioactivates carcinogens. Human dehydroepiandrosterone sulfotransferase (hSULT2A1) plays important biological roles by sulfating endogenous hydroxysteroids and exogenous xenobiotics. Genistein, mainly existing in soy food products, is a naturally occurring phytoestrogen with both chemopreventive and chemotherapeutic potential. Our previous studies have shown that genistein significantly induces hSULT2A1 in Hep G2 and Caco-2 cells. In this study, we investigated the roles of liver X receptor (LXRα) in the genistein induction of hSULT2A1. LXRs have been shown to induce expression of mouse Sult2a9 and hSULT2A1 gene. Our results demonstrate that LXRα mediates the genistein induction of hSULT2A1, supported by Western blot analysis results, hSULT2A1 promoter driven luciferase reporter gene assay results, and mRNA interference results. Chromatin immunoprecipitation (ChIP) assay results demonstrate that genistein increase the recruitment of hLXRα binding to the hSULT2A1 promoter. These results suggest that hLXRα plays an important role in the hSULT2A1 gene regulation. The biological functions of phytoestrogens may partially relate to their induction activity toward hydroxysteroid SULT. - Highlights: ► Liver X receptor α mediated genistein induction of hSULT2A1 in Hep G2 cells. ► LXRα and RXRα dimerization further activated this induction. ► Western blot results agreed well with luciferase reporter gene assay results. ► LXRs gene silencing significantly decreased hSULT2A1 expression. ► ChIP analysis suggested that genistein enhances hLXRα binding to the hSULT2A1 promoter.

  7. Increased levels of insulin and insulin-like growth factor-1 hybrid receptors and decreased glycosylation of the insulin receptor alpha- and beta-subunits in scrapie-infected neuroblastoma N2a cells.

    PubMed

    Nielsen, Daniel; Gyllberg, Hanna; Ostlund, Pernilla; Bergman, Tomas; Bedecs, Katarina

    2004-06-01

    We have previously shown that ScN2a cells (scrapie-infected neuroblastoma N2a cells) express 2-fold- and 4-fold-increased levels of IR (insulin receptor) and IGF-1R (insulin-like growth factor-1 receptor) respectively. In addition, the IR alpha- and beta-subunits are aberrantly processed, with apparent molecular masses of 128 and 85 kDa respectively, as compared with 136 and 95 kDa in uninfected N2a cells. Despite the 2-fold increase in IR protein, the number of (125)I-insulin-binding sites was slightly decreased in ScN2a cells [Ostlund, Lindegren, Pettersson and Bedecs (2001) Brain Res. 97, 161-170]. In order to determine the cellular localization of IR in ScN2a cells, surface biotinylation was performed, showing a correct IR trafficking and localization to the cell surface. The present study shows for the first time that neuroblastoma N2a cells express significant levels of IR-IGF-1R hybrid receptors, and in ScN2a cells the number of hybrid receptors was 2-fold higher than that found in N2a cells, potentially explaining the apparent loss of insulin-binding sites due to a lower affinity for insulin compared with the homotypic IR. Furthermore, the decreased molecular mass of IR subunits in ScN2a cells is not caused by altered phosphorylation or proteolytic processing, but rather by altered glycosylation. Enzymic deglycosylation of immunoprecipitated IR from N2a and ScN2a cells with endoglycosidase H, peptide N-glycosidase F and neuraminidase all resulted in subunits with increased electrophoretic mobility; however, the 8-10 kDa shift remained. Combined enzymic or chemical deglycosylation using anhydrous trifluoromethane sulphonic acid treatment ultimately showed that the IR alpha- and beta-subunits from ScN2a cells are aberrantly glycosylated. The increased formation of IR-IGF-1R hybrids in ScN2a cells may be part of a neuroprotective response to prion infection. The degree and functional significance of aberrantly glycosylated proteins in ScN2a cells remain to be

  8. Analysis of variations in the glutamate receptor, N-methyl D-aspartate 2A (GRIN2A) gene reveals their relative importance as genetic susceptibility factors for heroin addiction.

    PubMed

    Zhao, Bin; Zhu, Yongsheng; Wang, Wei; Cui, Hai-Min; Wang, Yun-Peng; Lai, Jiang-Hua

    2013-01-01

    The glutamate receptor, N-methyl D-aspartate 2A (GRIN2A) gene that encodes the 2A subunit of the N-methyl D-aspartate (NMDA) receptor was recently shown to be involved in the development of opiate addiction. Genetic polymorphisms in GRIN2A have a plausible role in modulating the risk of heroin addiction. An association of GRIN2A single-nucleotide polymorphisms (SNPs) with heroin addiction was found earlier in African Americans. To identify markers that contribute to the genetic susceptibility to heroin addiction, we examined the potential association between heroin addiction and forty polymorphisms of the GRIN2A gene using the MassARRAY system and GeneScan in this study. The frequency of the (GT)26 repeats (rs3219790) in the heroin addiction group was significantly higher than that in the control group (χ(2) = 5.360, P = 0.021). The allele frequencies of three polymorphisms (rs1102972, rs1650420, and rs3104703 in intron 3) were strongly associated with heroin addiction (P<0.001, 0.0002, and <0.001, after Bonferroni correction). Three additional SNPs from the same intron (rs1071502, rs6497730, and rs1070487) had nominally significant P values for association (P<0.05), but did not pass the threshold value. Haplotype analysis revealed that the G-C-T-C-C-T-A (block 6) and T-T (block 10) haplotypes of the GRIN2A gene displayed a protective effect (P = <0.001 and 0.003). These findings point to a role for GRIN2A polymorphisms in heroin addiction among the Han Chinese from Shaanxi province, and may be informative for future genetic or neurobiological studies on heroin addiction.

  9. Autoantibodies to NR2A Peptide of the Glutamate/NMDA Receptor in Patients with Seizure Disorders in Neuropsychiatric Systemic Lupus Erythematosus

    PubMed Central

    Wang, Xue-er; Mei, Qing-hua; Jiang, Wen-qing

    2017-01-01

    Objective. Seizure disorders are one of the most disabling, life-threatening, and the least understood syndromes associated with neuropsychiatric SLE (NPSLE). N-Methyl-D-aspartate (NMDA) receptors are a subgroup of the glutamate receptor family, whose NR2A subunit was found on neuronal cells (anti-NR2A) in NPSLE patients with different types of epilepsy. The present study was conducted to determine the serum levels of anti-NR2A antibodies in a large group of SLE patients, to investigate the possible correlation between the presence of the NR2A specific antibodies and NPSLE-related seizure disorders. Methods and Results. The study population consisted of 107 SLE patients and 43 age- and sex-matched healthy controls. 73 SLE patients had active disease. 36 of these had NPSLE. NMDA levels were measured by ELISA. Clinical and serological parameters were assessed according to routine procedures. The levels of anti-NR2A antibodies were significantly higher in NPSLE patients, compared with non-NPSLE patients and healthy controls. Furthermore, the levels of NPSLE in patients with seizure disorders were shown to be higher than in those with cognitive dysfunction and other CNS symptoms, however, without significance. Increase in serum anti-NR2A antibodies levels correlated to anti-dsDNA antibody and SLEDAI as well as complement levels. Conclusion. We suggest that anti-NR2A antibodies play a role in the pathogenesis of NPSLE with seizure disorders. PMID:28154472

  10. The 5-hydroxytryptamine2A receptor is involved in (+)-norfenfluramine-induced arterial contraction and blood pressure increase in deoxycorticosterone acetate-salt hypertension.

    PubMed

    Ni, Wei; Fink, Gregory D; Watts, Stephanie W

    2007-05-01

    The highly effective anorexigen (+)-fenfluramine was widely used to control body weight until the association with primary pulmonary hypertension and valvular heart disease. (+)-Norfenfluramine is the major hepatic metabolite of (+)-fenfluramine and is primarily responsible for the anorexic effect as well as side effects. We reported that (+)-norfenfluramine causes vasoconstriction and a blood pressure increase in rats with normal blood pressure via the 5-hydroxytryptamine (5-HT)2A receptor. With the knowledge that (+)-norfenfluramine also has affinity for 5-HT2B receptors and that arterial 5-HT2B receptor expression is up-regulated in deoxycorticosterone acetate (DOCA)-salt hypertension, we tested the hypothesis that (+)-norfenfluramine-induced vasoconstriction and pressor effects are potentiated in DOCA-salt hypertensive rats in a 5-HT2 receptor-dependent manner. Contractions of arteries were measured using an isolated tissue bath system or myograph. Mean arterial blood pressure was measured in chronically instrumented conscious rats. Effects of (+)-norfenfluramine in stimulating arterial contraction (leftward shift versus SHAM, aorta, 5.13-fold; renal artery, 1.95-fold; mesenteric resistance artery, 1.77-fold) and raising blood pressure were significantly enhanced in hypertension. In arteries from both normotensive and hypertensive rats, (+)-norfenfluramine-induced contraction in aorta was inhibited by 5-HT2A receptor antagonists, ketanserin and LY53857 (4-isopropyl-7-methyl-9-(2-hydroxy-1-meth ylpropoxycarbonyl)4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline), but not by the 5-HT2B receptor antagonist, LY272015 [6-chloro-5-methyl-N-(5-quinolinyl)-2,3-dihydro-1H-indole-1-carboxamide]. Ketanserin (3 mg/kg) reduced (+)-norfenfluramine-induced pressor response in both SHAM and DOCA rats. Our results demonstrate that (+)-norfenfluramine-induced arterial contraction and blood pressure increases are potentiated in DOCA-salt hypertensive rats. However, it is the 5

  11. Cerebral metabolic responses to 5-HT2A/C receptor activation in mice with genetically modified serotonin transporter (SERT) expression.

    PubMed

    Dawson, Neil; Ferrington, Linda; Lesch, Klaus-Peter; Kelly, Paul A T

    2011-01-01

    Variation in the human serotonin transporter gene (hSERT; 5-HTT) resulting in a life-long alteration in SERT function influences anxiety and the risk of developing affective disorders. The mechanisms underlying the influence of the hSERT gene on these phenotypes remain unclear but may involve altered 5-HT receptor function. Here we characterise the cerebral metabolic response to 5-HT(2A/C) receptor activation in two transgenic mouse models of altered SERT function, SERT knock-out (SERT KO) and hSERT over-expressing (hSERT OE) mice, to test the hypothesis that genetically mediated variability in SERT expression alters 5-HT(2A/C) function. We found that a constitutive increase in SERT expression (hSERT OE) enhanced, whereas a constitutive decrease in SERT expression (SERT KO) attenuated, 5-HT(2A/C) function. Therefore, altered 5-HT(2A/C) receptor functioning in response to hSERT gene variation may contribute to its influence on affective phenotypes.

  12. Protein kinase A mediates adenosine A2a receptor modulation of neurotransmitter release via synapsin I phosphorylation in cultured cells from medulla oblongata.

    PubMed

    Matsumoto, Joao Paulo Pontes; Almeida, Marina Gomes; Castilho-Martins, Emerson Augusto; Costa, Maisa Aparecida; Fior-Chadi, Debora Rejane

    2014-08-01

    Synaptic transmission is an essential process for neuron physiology. Such process is enabled in part due to modulation of neurotransmitter release. Adenosine is a synaptic modulator of neurotransmitter release in the Central Nervous System, including neurons of medulla oblongata, where several nuclei are involved with neurovegetative reflexes. Adenosine modulates different neurotransmitter systems in medulla oblongata, specially glutamate and noradrenaline in the nucleus tractussolitarii, which are involved in hypotensive responses. However, the intracellular mechanisms involved in this modulation remain unknown. The adenosine A2a receptor modulates neurotransmitter release by activating two cAMP protein effectors, the protein kinase A and the exchange protein activated by cAMP. Therefore, an in vitro approach (cultured cells) was carried out to evaluate modulation of neurotransmission by adenosine A2a receptor and the signaling intracellular pathway involved. Results show that the adenosine A2a receptor agonist, CGS 21680, increases neurotransmitter release, in particular, glutamate and noradrenaline and such response is mediated by protein kinase A activation, which in turn increased synapsin I phosphorylation. This suggests a mechanism of A2aR modulation of neurotransmitter release in cultured cells from medulla oblongata of Wistar rats and suggest that protein kinase A mediates this modulation of neurotransmitter release via synapsin I phosphorylation.

  13. Escitalopram reduces attentional performance in anxious older adults with high-expression genetic variants at serotonin 2A and 1B receptors

    PubMed Central

    Lenze, Eric J.; Dixon, David; Nowotny, Petra; Lotrich, Francis E.; Doré, Peter M.; Pollock, Bruce G.; Hinrichs, Anthony L.; Butters, Meryl A.

    2014-01-01

    Older adults are among the most vulnerable to adverse cognitive effects of psychotropic medications and, therefore, the personalization of psychotropic treatment based on adverse drug reactions in this demographic is of great importance. We examined changes on neuropsychological tests of attention attributable to selective serotonin reuptake inhibitor (SSRI) treatment in anxious older adults. We also examined whether variation in serotonin receptor genes was associated with reduced attentional performance with SSRIs. We examined change from pre- to post-treatment in two attention measures – digit span and coding – in 133 adults aged ≥60 yr with generalized anxiety disorder in a 12-wk trial of escitalopram vs. placebo. We also examined attentional change in relation to genetic variability in four central serotonin receptors: the serotonin transporter and serotonin 1A, 2A and 1B receptors. Digit span scores were significantly lowered in patients receiving escitalopram relative to placebo, indicating reduced attentional performance attributable to the SSRI. Individuals with high-transcription variants in the receptors 5-HTR2A rs6311 and 5-HTR1B rs11568817 had greater reductions in attention with SSRI treatment compared to placebo. We conclude that SSRIs reduce attention in older adults, particularly in those with high-expression genetic variants at the serotonin 2A and 1B receptors. Analysing neuropsychological changes with SSRIs in relation to genetic variation in the serotonin system may be a useful strategy for detecting subgroups of older adults who are more susceptible to side-effects of SSRIs. These results, if confirmed, could lead to the personalization of SSRI use to reduce adverse neurocognitive effects. PMID:22717018

  14. Escitalopram reduces attentional performance in anxious older adults with high-expression genetic variants at serotonin 2A and 1B receptors.

    PubMed

    Lenze, Eric J; Dixon, David; Nowotny, Petra; Lotrich, Francis E; Doré, Peter M; Pollock, Bruce G; Hinrichs, Anthony L; Butters, Meryl A

    2013-03-01

    Older adults are among the most vulnerable to adverse cognitive effects of psychotropic medications and, therefore, the personalization of psychotropic treatment based on adverse drug reactions in this demographic is of great importance. We examined changes on neuropsychological tests of attention attributable to selective serotonin reuptake inhibitor (SSRI) treatment in anxious older adults. We also examined whether variation in serotonin receptor genes was associated with reduced attentional performance with SSRIs. We examined change from pre- to post-treatment in two attention measures - digit span and coding - in 133 adults aged ≥60 yr with generalized anxiety disorder in a 12-wk trial of escitalopram vs. placebo. We also examined attentional change in relation to genetic variability in four central serotonin receptors: the serotonin transporter and serotonin 1A, 2A and 1B receptors. Digit span scores were significantly lowered in patients receiving escitalopram relative to placebo, indicating reduced attentional performance attributable to the SSRI. Individuals with high-transcription variants in the receptors 5-HTR2A rs6311 and 5-HTR1B rs11568817 had greater reductions in attention with SSRI treatment compared to placebo. We conclude that SSRIs reduce attention in older adults, particularly in those with high-expression genetic variants at the serotonin 2A and 1B receptors. Analysing neuropsychological changes with SSRIs in relation to genetic variation in the serotonin system may be a useful strategy for detecting subgroups of older adults who are more susceptible to side-effects of SSRIs. These results, if confirmed, could lead to the personalization of SSRI use to reduce adverse neurocognitive effects.

  15. A Model of Post-Infection Fatigue Is Associated with Increased TNF and 5-HT2A Receptor Expression in Mice

    PubMed Central

    Couch, Yvonne; Xie, Qin; Lundberg, Louise; Sharp, Trevor; Anthony, Daniel C.

    2015-01-01

    It is well documented that serotonin (5-HT) plays an important role in psychiatric illness. For example, myalgic encephalomyelitis (ME/CFS), which is often provoked by infection, is a disabling illness with an unknown aetiology and diagnosis is based on symptom-specific criteria. However, 5-HT2A receptor expression and peripheral cytokines are known to be upregulated in ME. We sought to examine the relationship between the 5-HT system and cytokine expression following systemic bacterial endotoxin challenge (LPS, 0.5mg/kg i.p.), at a time when the acute sickness behaviours have largely resolved. At 24 hours post-injection mice exhibit no overt changes in locomotor behaviour, but do show increased immobility in a forced swim test, as well as decreased sucrose preference and reduced marble burying activity, indicating a depressive-like state. While peripheral IDO activity was increased after LPS challenge, central activity levels remained stable and there was no change in total brain 5-HT levels or 5-HIAA/5-HT. However, within the brain, levels of TNF and 5-HT2A receptor mRNA within various regions increased significantly. This increase in receptor expression is reflected by an increase in the functional response of the 5-HT2A receptor to agonist, DOI. These data suggest that regulation of fatigue and depressive-like moods after episodes of systemic inflammation may be regulated by changes in 5-HT receptor expression, rather than by levels of enzyme activity or cytokine expression in the CNS. PMID:26147001

  16. Horse chestnut extract contracts bovine vessels and affects human platelet aggregation through 5-HT(2A) receptors: an in vitro study.

    PubMed

    Felixsson, Emma; Persson, Ingrid A-L; Eriksson, Andreas C; Persson, Karin

    2010-09-01

    Extract from seeds and bark of horse chestnut (Aesculus hippocastanum L) is used as an herbal medicine against chronic venous insufficiency. The effect and mechanism of action on veins, arteries, and platelets are not fully understood. The aim of this study was to investigate the effects and mechanisms of action of horse chestnut on the contraction of bovine mesenteric veins and arteries, and human platelet aggregation. Contraction studies showed that horse chestnut extract dose-dependently contracted both veins and arteries, with the veins being the most sensitive. Contraction of both veins and arteries were significantly inhibited by the 5-HT(2A) receptor antagonist ketanserin. No effect on contraction was seen with the cyclooxygenase inhibitor indomethacin, the alpha(1) receptor antagonist prazosin or the angiotensin AT(1) receptor antagonist saralasin neither in veins nor arteries. ADP-induced human platelet aggregation was significantly reduced by horse chestnut. A further reduction was seen with the extract in the presence of ketanserin. In conclusion, horse chestnut contraction of both veins and arteries is, at least partly, mediated through 5-HT(2A) receptors. Human platelet aggregation is reduced by horse chestnut. The clinical importance of these findings concerning clinical use, possible adverse effects, and drug interactions remains to be investigated.

  17. Regional distribution and behavioral correlates of 5-HT(2A) receptors in Alzheimer's disease with [(18)F]deuteroaltanserin and PET.

    PubMed

    Santhosh, Lekshmi; Estok, Kristina M; Vogel, Rebecca S; Tamagnan, Gilles D; Baldwin, Ronald M; Mitsis, Effie M; Macavoy, Martha G; Staley, Julie K; van Dyck, Christopher H

    2009-09-30

    Postmortem studies show reductions in brain serotonin 2A (5-HT(2A)) receptors in Alzheimer's disease (AD). Converging evidence also suggests that serotonergic dysregulation may contribute to behavioral symptoms that frequently occur in AD. This study aimed to define regional reductions in 5-HT(2A) binding in AD patients and to examine their behavioral correlates. Nine patients with probable AD and eight elderly controls were studied using a constant infusion paradigm for equilibrium modeling of [(18)F]deuteroaltanserin with positron emission tomography (PET). Region of interest analyses were performed on PET images coregistered to MRI scans. The outcome measures BP(P) (ratio of specific brain uptake to total plasma parent concentration) and BP(ND) (ratio of specific to nondisplaceable uptake) were obtained for pertinent cortical and subcortical regions. AD patients showed a statistically significant decrease in the anterior cingulate in both BP(P) and BP(ND), but in no other region. Within the AD patient sample, no significant correlations were observed between regional 5-HT(2A) binding and behavioral measures, including depressive and psychotic symptoms. These results confirm a reduction in cortical 5-HT(2A) receptors in AD, specifically in the anterior cingulate. However, in a limited AD patient sample, they fail to demonstrate a relationship between regional 5-HT(2A) binding and major behavioral symptoms.

  18. Involvement of local serotonin-2A but not serotonin-1B receptors in the reinforcing effects of ethanol within the posterior ventral tegmental area of female Wistar rats

    PubMed Central

    Ding, Zheng-Ming; Toalston, Jamie E.; Oster, Scott M.; McBride, William J.; Rodd, Zachary A.

    2010-01-01

    Rationale Previous studies indicated that ethanol could be self-infused into the posterior ventral tegmental area (p-VTA) and that activation of local serotonin-3 (5-HT3) receptors was involved. 5-HT1B and 5-HT2A receptors are involved in the effects of 5-HT and ethanol on VTA dopamine neurons. Objective The current study used the intracranial self-administration (ICSA) procedure to determine the involvement of local 5-HT1B and 5-HT2A receptors in the self-infusion of ethanol into the p-VTA. Materials and methods Female Wistar rats were implanted unilaterally with a guide cannula aimed at the p-VTA. Seven days after surgery, rats were placed into the two-lever operant conditioning chambers for ICSA tests. The tests consisted of four acquisition sessions with self-infusion of 200 mg% ethanol alone, two or three sessions with co-infusion of the 5-HT1B antagonist GR 55562 (10, 100, or 200 μM) or the 5-HT2A antagonist R-96544 (10, 100, or 200 μM) with 200 mg% ethanol, and one final session with 200 mg% ethanol alone. Results During the acquisition sessions, all rats readily self-infused ethanol and discriminated the active from inactive lever. Co-infusion of GR 55562, at all three doses, had no effect on the self-infusion of ethanol. In contrast, co-infusion of R-96544, at the two higher doses, attenuated responding on the active lever for ethanol infusion (p<0.05). Conclusion The results suggest that the reinforcing effects of ethanol within the p-VTA are modulated, at least in part, by activation of local 5-HT2A, but not 5-HT1B, receptors. PMID:19165471

  19. The adenosine A2A receptor antagonist ZM241385 enhances neuronal survival after oxygen-glucose deprivation in rat CA1 hippocampal slices

    PubMed Central

    Pugliese, AM; Traini, C; Cipriani, S; Gianfriddo, M; Mello, T; Giovannini, MG; Galli, A; Pedata, F

    2009-01-01

    Background and purpose: Activation of adenosine A2A receptors in the CA1 region of rat hippocampal slices during oxygen-glucose deprivation (OGD), a model of cerebral ischaemia, was investigated. Experimental approach: We made extracellular recordings of CA1 field excitatory postsynaptic potentials (fepsps) followed by histochemical and immunohistochemical techniques coupled to Western blots. Key results: OGD (7 or 30 min duration) elicited an irreversible loss of fepsps invariably followed by the appearance of anoxic depolarization (AD), an unambiguous sign of neuronal damage. The application of the selective adenosine A2A receptor antagonist, ZM241385 (4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-ylamino]ethyl)phenol; 100–500 nmol·L−1) prevented or delayed AD appearance induced by 7 or 30 min OGD and protected from the irreversible fepsp depression elicited by 7 min OGD. Two different selective adenosine A2A receptor antagonists, SCH58261 and SCH442416, were less effective than ZM241385 during 7 min OGD. The extent of CA1 cell injury was assessed 3 h after the end of 7 min OGD by propidium iodide. Substantial CA1 pyramidal neuronal damage occurred in untreated slices, exposed to OGD, whereas injury was significantly prevented by 100 nmol·L−1 ZM241385. Glial fibrillary acid protein (GFAP) immunostaining showed that 3 h after 7 min OGD, astrogliosis was appreciable. Western blot analysis indicated an increase in GFAP 30 kDa fragment which was significantly reduced by treatment with 100 nmol·L−1 ZM241385. Conclusions and implications: In the CA1 hippocampus, antagonism of A2A adenosine receptors by ZM241385 was protective during OGD (a model of cerebral ischaemia) by delaying AD appearance, decreasing astrocyte activation and improving neuronal survival. PMID:19422385

  20. Repeated administration of Yokukansan inhibits DOI-induced head-twitch response and decreases expression of 5-hydroxytryptamine (5-HT)2A receptors in the prefrontal cortex.

    PubMed

    Egashira, Nobuaki; Iwasaki, Katsunori; Ishibashi, Ayumi; Hayakawa, Kazuhide; Okuno, Ryoko; Abe, Moe; Uchida, Naoki; Mishima, Kenichi; Takasaki, Kotaro; Nishimura, Ryoji; Oishi, Ryozo; Fujiwara, Michihiro

    2008-08-01

    Behavioral and psychological symptoms of dementia (BPSD) are commonly seen in patients with Alzheimer's disease (AD) and other forms of senile dementia. BPSD have a serious impact on the quality of life of dementia patients, as well as their caregivers. However, an effective drug therapy for BPSD has not been established. Recently, the traditional Japanese medicine Yokukansan (YKS, Yi-gan san in Chinese) has been reported to improve BPSD in a randomized, single-blind, placebo-controlled study. Moreover, abnormalities of the serotonin (5-HT) system such as 5-HT2A receptors have been reported to be associated with BPSD of AD patients. In the present study, we investigated the effect of YKS on head-twitch response induced by 2,5-dimethoxy-4-iodoamphetamine (DOI, 5 mg/kg, i.p.) in mice, a behavioral response that is mediated, in part, by 5-HT2A receptors. Acute treatment with YKS (100 and 300 mg/kg, p.o.) had no effect on the DOI-induced head-twitch response, whilst 14 days repeated treatment with YKS (300 mg/kg, p.o.) significantly inhibited this response. Moreover, repeated treatment with YKS (300 mg/kg, p.o.) decreased expression of 5-HT2A receptors in the prefrontal cortex, which is part of the circuitry mediating the head-twitch response. These findings suggest that the inhibition of DOI-induced head-twitch response by YKS may be mediated, in part, by altered expression of 5-HT2A receptors in the prefrontal cortex, which suggests the involvement of the 5-HT system in psychopharmacological effects of YKS.

  1. Discovery of GluN2A-Selective NMDA Receptor Positive Allosteric Modulators (PAMs): Tuning Deactivation Kinetics via Structure-Based Design.

    PubMed

    Volgraf, Matthew; Sellers, Benjamin D; Jiang, Yu; Wu, Guosheng; Ly, Cuong Q; Villemure, Elisia; Pastor, Richard M; Yuen, Po-wai; Lu, Aijun; Luo, Xifeng; Liu, Mingcui; Zhang, Shun; Sun, Liang; Fu, Yuhong; Lupardus, Patrick J; Wallweber, Heidi J A; Liederer, Bianca M; Deshmukh, Gauri; Plise, Emile; Tay, Suzanne; Reynen, Paul; Herrington, James; Gustafson, Amy; Liu, Yichin; Dirksen, Akim; Dietz, Matthias G A; Liu, Yanzhou; Wang, Tzu-Ming; Hanson, Jesse E; Hackos, David; Scearce-Levie, Kimberly; Schwarz, Jacob B

    2016-03-24

    The N-methyl-D-aspartate receptor (NMDAR) is a Na(+) and Ca(2+) permeable ionotropic glutamate receptor that is activated by the coagonists glycine and glutamate. NMDARs are critical to synaptic signaling and plasticity, and their dysfunction has been implicated in a number of neurological disorders, including schizophrenia, depression, and Alzheimer's disease. Herein we describe the discovery of potent GluN2A-selective NMDAR positive allosteric modulators (PAMs) starting from a high-throughput screening hit. Using structure-based design, we sought to increase potency at the GluN2A subtype, while improving selectivity against related α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). The structure-activity relationship of channel deactivation kinetics was studied using a combination of electrophysiology and protein crystallography. Effective incorporation of these strategies resulted in the discovery of GNE-0723 (46), a highly potent and brain penetrant GluN2A-selective NMDAR PAM suitable for in vivo characterization.

  2. Differences in the C-terminus contribute to variations in trafficking between rat and human 5-HT(2A) receptor isoforms: identification of a primate-specific tripeptide ASK motif that confers GRK-2 and beta arrestin-2 interactions.

    PubMed

    Bhattacharya, Aditi; Sankar, Shobhana; Panicker, Mitradas M

    2010-02-01

    Internalization and recycling of G-protein coupled receptors are important cellular processes regulating receptor function. These are receptor-subtype and cell type-specific. Although important, trafficking variations between receptor isoforms of different species has received limited attention. We report here, differences in internalization and recycling between rat and human serotonin 2A receptor (5-HT(2A)R) isoforms expressed in human embryonic kidney 293 cells in response to serotonin. Although the human and rat 5-HT(2A)Rs differ by only a few amino acids, the human receptor takes longer to recycle to the cell surface after internalization, with the additional involvement of beta arrestin-2 and G-protein receptor kinase 2. The interaction of beta arrestin-2 with the human receptor causes the delay in recycling and is dependent on a primate-specific ASK motif present in the C-terminus of the receptor. Conversion of this motif to NCT, the corresponding sequence present in the rat isoform, results in the human isoform trafficking like the rat receptor. Replacing the serine 457 with alanine in the ASK motif of human isoform resulted in faster recycling, although with continued arrestin-dependent internalization. This study establishes significant differences between the two isoforms with important implications in our understanding of the human 5-HT(2A)R functions; and indicates that extrapolating results from non-human receptor isoforms to human subtypes is not without caveats.

  3. Effects of the serotonin 5-HT2A and 5-HT2C receptor ligands on the discriminative stimulus effects of nicotine in rats.

    PubMed

    Zaniewska, Magdalena; McCreary, Andrew C; Przegaliński, Edmund; Filip, Malgorzata

    2007-10-01

    The present study tested the hypothesis that serotonergic (5-HT) 5-HT2A or 5-HT2C receptors or their pharmacological stimulation modulated the discriminative stimulus effects of nicotine in male Wistar rats. To this end the selective 5-HT2A receptor antagonist R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol (M100,907; 0.5-1 mg/kg, i.p.), the functional 5-HT2A receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI; 0.1-1 mg/kg, s.c.), the selective 5-HT2C receptor antagonist 6-chloro-5-methyl-1-{[2-(2-methylpyrid-3-yloxy)pyrid-5-yl]carbamoyl}indoline (SB 242,084; 0.25-1 mg/kg, i.p.) and the 5-HT2C receptor agonists (S)-2-chloro-5-fluoro-indol-1-yl)-1-methylethylamine fumarate (Ro 60-0175; 0.3-1 mg/kg, s.c.) and (7bR, 10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole (WAY 163,909; 0.75-1.5 mg/kg, i.p.) were used. Additionally, the effects of the selective alpha4beta2 nicotinic acetylcholine receptor subtype agonist 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-IA; 0.01 mg/kg, s.c.) were investigated. In rats trained to discriminate (-)-nicotine (0.4 mg/kg, s.c.) from saline in a two-lever, water-reinforced fixed ratio 10 task, substitutions were not observed with 5-HT2 receptor ligands (<32% nicotine-lever responding), conversely 5-IA induced a full substitution (100% nicotine-lever responding). In combination studies, fixed doses of M100,907 (0.5-1 mg/kg) or SB 242,084 (0.25-1 mg/kg) did not alter the dose-response curve of nicotine, while DOI (0.3 mg/kg), Ro 60-0175 (1 mg/kg) and WAY 163,909 (1 and 1.5 mg/kg) attenuated the discriminative stimulus effects of nicotine. The decrease in the expression of the discriminative stimulus effects of nicotine produced by DOI was blocked by M100,907 (1 mg/kg), but not by SB 242,084 (1 mg/kg), while that evoked by Ro 60-0175 or WAY 163,909 was blocked by SB 242,084 (1 mg/kg), but not by M100,907 (1 mg/kg). Further studies showed that

  4. Differential contribution of the NR1- and NR2A-subunits to the selectivity filter of recombinant NMDA receptor channels.

    PubMed Central

    Wollmuth, L P; Kuner, T; Seeburg, P H; Sakmann, B

    1996-01-01

    1. The molecular determinants for the narrow constriction of recombinant N-methyl-D-aspartate (NMDA) receptor channels composed of wild-type and mutant NR1- and NR2A-subunits were studied in Xenopus oocytes. 2. The relative permeability of differently sized organic cations was used as an indicator of the size of the narrow constriction. From measured reversal potentials under bi-ionic conditions with K+ as the reference solution, permeability ratios were calculated with the Lewis equation. 3. For wild-type NMDA receptor channels, five organic cations showed clear reversal potentials, with permeability ratios (PX/PK): ammonium, 1.28; methylammonium, 0.48; dimethylammonium (DMA), 0.20; diethylammonium, 0.07; and dimethylethanol-ammonium, 0.02. 4. Mutation of the N-site asparagine (N) to glutamine (Q) at homologous positions in either NR1 (position 598) or NR2A (position 595) increased the permeability of DMA relative to wild-type channels about equally. However, for larger sized organic cations, the NR1(N598Q) mutation had stronger effects on increasing their permeability whereas the NR2A(N595Q) mutation was without effect. These changes in organic cation permeability suggest that the NR1(N598Q) mutation increases the pore size while the NR2A(N595Q) mutation does not. 5. Channels in which the NR1 N-site asparagine was replaced by the smaller glycine (G), NR1(N598G)-NR2A, showed the largest increase in pore size of all sites examined in either subunit. In contrast, in the NR2A-subunit the same N-site substitution to glycine produced only small effects on pore size. 6. For the NR2A-subunit, an asparagine residue (position 596) on the C-terminal side of the N-site, when mutated to larger or smaller sized amino acids, produced large, volume-specific effects on pore size. The mutant channel NR1-NR2A(N596G) had the largest increase in pore size of all sites examined in the NR2A-subunit. In contrast, mutation of the homologous position in the NR1-subunit had no effect on

  5. The Brassinosteroid-Activated BRI1 Receptor Kinase Is Switched off by Dephosphorylation Mediated by Cytoplasm-Localized PP2A B' Subunits.

    PubMed

    Wang, Ruiju; Liu, Mengmeng; Yuan, Min; Oses-Prieto, Juan A; Cai, Xingbo; Sun, Ying; Burlingame, Alma L; Wang, Zhi-Yong; Tang, Wenqiang

    2016-01-04

    Brassinosteroid (BR) binding activates the receptor kinase BRI1 by inducing heterodimerization with its co-receptor kinase BAK1; however, the mechanisms that reversibly inactivate BRI1 remain unclear. Here we show that cytoplasm-localized protein phosphatase 2A (PP2A) B' regulatory subunits interact with BRI1 to mediate its dephosphorylation and inactivation. Loss-of-function and overexpression experiments showed that a group of PP2A B' regulatory subunits, represented by B'η, negatively regulate BR signaling by decreasing BRI1 phosphorylation. BR increases the expression levels of these B' subunits, and B'η interacts preferentially with phosphorylated BRI1, suggesting that the dynamics of BR signaling are modulated by the PP2A-mediated feedback inactivation of BRI1. Compared with PP2A B'α and B'β, which promote BR responses by dephosphorylating the downstream transcription factor BZR1, the BRI1-inactivating B' subunits showed similar binding to BRI1 and BZR1 but distinct subcellular localization. Alteration of the nuclear/cytoplasmic localization of the B' subunits revealed that cytoplasmic PP2A dephosphorylates BRI1 and inhibits the BR response, whereas nuclear PP2A dephosphorylates BZR1 and activates the BR response. Our findings not only identify the PP2A regulatory B subunits that mediate the binding and dephosphorylation of BRI1, but also demonstrate that the subcellular localization of PP2A specifies its substrate selection and distinct effects on BR signaling.

  6. Activation of Th1 and Tc1 cell adenosine A2A receptors directly inhibits IL-2 secretion in vitro and IL-2-driven expansion in vivo.

    PubMed

    Erdmann, Andreas A; Gao, Zhan-Guo; Jung, Unsu; Foley, Jason; Borenstein, Todd; Jacobson, Kenneth A; Fowler, Daniel H

    2005-06-15

    To evaluate the direct effect of adenosine on cytokine-polarized effector T cells, murine type 1 helper T cells (Th1) and type 1 cytotoxic T lymphocytes (Tc1) and Th2/Tc2 cells were generated using an antigen-presenting cell (APC)-free method. Tc1 and Tc2 cells had similar adenosine signaling, as measured by intracellular cyclic AMP (cAMP) increase upon adenosine A(2A) receptor agonism by CGS21680 (CGS). CGS greatly reduced Tc1 and Tc2 cell interleukin 2 (IL-2) and tumor necrosis factor alpha (TNF-alpha) secretion, with nominal effect on interferon gamma (IFN-gamma) secretion. Tc2 cell IL-4 and IL-5 secretion was not reduced by CGS, and IL-10 secretion was moderately reduced. Agonist-mediated inhibition of IL-2 and TNF-alpha secretion occurred via A(2A) receptors, with no involvement of A(1), A(2B), or A(3) receptors. Adenosine agonist concentrations that abrogated cytokine secretion did not inhibit Tc1 or Tc2 cell cytolytic function. Adenosine modulated effector T cells in vivo, as CGS administration reduced CD4(+)Th1 and CD8(+)Tc1 cell expansion to alloantigen and, in a separate model, reduced antigen-specific CD4(+) Th1 cell numbers. Remarkably, agonist-mediated T-cell inhibition was abrogated by in vivo IL-2 therapy. Adenosine receptor activation therefore preferentially inhibits type I cytokine secretion, most notably IL-2. Modulation of adenosine receptors may thus represent a suitable target primarily for inflammatory conditions mediated by Th1 and Tc1 cells.

  7. Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors.

    PubMed

    Viñals, Xavier; Moreno, Estefanía; Lanfumey, Laurence; Cordomí, Arnau; Pastor, Antoni; de La Torre, Rafael; Gasperini, Paola; Navarro, Gemma; Howell, Lesley A; Pardo, Leonardo; Lluís, Carmen; Canela, Enric I; McCormick, Peter J; Maldonado, Rafael; Robledo, Patricia

    2015-07-01

    Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties.

  8. Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells.

    PubMed

    Algarni, Alanood S; Hargreaves, Alan J; Dickenson, John M

    2017-03-15

    The PAC1 receptor and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 activity by the PAC1 receptor in retinoic acid-induced differentiating N2a neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. TG2 phosphorylation was monitored via immunoprecipitation and Western blotting. The role of TG2 in PAC1 receptor-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27). PACAP-27 mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON and R283 and by pharmacological inhibition of protein kinase A (KT 5720 and Rp-cAMPs), protein kinase C (Ro 31-8220), MEK1/2 (PD 98059), and removal of extracellular Ca(2+). Fluorescence microscopy demonstrated PACAP-27 induced in situ TG2 activity. TG2 inhibition blocked PACAP-27 induced attenuation of hypoxia-induced cell death and outgrowth of axon-like processes. TG2 activation and cytoprotection were also observed in human SH-SY5Y cells. Together, these results demonstrate that TG2 activity was stimulated downstream of the PAC1 receptor via a multi protein kinase dependent pathway. Furthermore, PAC1 receptor-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results highlight the importance of TG2 in the cellular functions of the PAC1 receptor.

  9. Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors

    PubMed Central

    Lanfumey, Laurence; Cordomí, Arnau; Pastor, Antoni; de La Torre, Rafael; Gasperini, Paola; Navarro, Gemma; Howell, Lesley A.; Pardo, Leonardo; Lluís, Carmen; Canela, Enric I.; McCormick, Peter J.; Maldonado, Rafael; Robledo, Patricia

    2015-01-01

    Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties. PMID:26158621

  10. Effects of the 5-HT2C receptor agonist Ro60-0175 and the 5-HT2A receptor antagonist M100907 on nicotine self-administration and reinstatement.

    PubMed

    Fletcher, Paul J; Rizos, Zoë; Noble, Kevin; Soko, Ashlie D; Silenieks, Leo B; Lê, Anh Dzung; Higgins, Guy A

    2012-06-01

    The reinforcing effects of nicotine are mediated in part by brain dopamine systems. Serotonin, acting via 5-HT(2A) and 5-HT(2C) receptors, modulates dopamine function. In these experiments we examined the effects of the 5-HT(2C) receptor agonist Ro60-0175 and the 5-HT(2A) receptor antagonist (M100907, volinanserin) on nicotine self-administration and reinstatement of nicotine-seeking. Male Long-Evans rats self-administered nicotine (0.03 mg/kg/infusion, IV) on either a FR5 or a progressive ratio schedule of reinforcement. Ro60-0175 reduced responding for nicotine on both schedules. While Ro60-0175 also reduced responding for food reinforcement, response rates under drug treatment were several-fold higher than in animals responding for nicotine. M100907 did not alter responding for nicotine, or food, on either schedule. In tests of reinstatement of nicotine-seeking, rats were first trained to lever press for IV infusions of nicotine; each infusion was also accompanied by a compound cue consisting of a light and tone. This response was then extinguished over multiple sessions. Injecting rats with a nicotine prime (0.15 mg/kg) reinstated responding; reinstatement was also observed when responses were accompanied by the nicotine associated cue. Ro60-0175 attenuated reinstatement of responding induced by nicotine and by the cue. The effects of Ro60-0175 on both forms of reinstatement were blocked by the 5-HT(2C) receptor antagonist SB242084. M100907 also reduced reinstatement induced by either the nicotine prime or by the nicotine associated cue. The results indicate that 5-HT(2C) and 5-HT(2A) receptors may be potential targets for therapies to treat some aspects of nicotine dependence.

  11. Genetic deletion of the adenosine A(2A) receptor prevents nicotine-induced upregulation of α7, but not α4β2* nicotinic acetylcholine receptor binding in the brain.

    PubMed

    Metaxas, Athanasios; Al-Hasani, Ream; Farshim, Pamela; Tubby, Kristina; Berwick, Amy; Ledent, Catherine; Hourani, Susanna; Kitchen, Ian; Bailey, Alexis

    2013-08-01

    Considerable evidence indicates that adenosine A(2A) receptors (A(2A)Rs) modulate cholinergic neurotransmission, nicotinic acetylcholine receptor (nAChR) function, and nicotine-induced behavioural effects. To explore the interaction between A(2A) and nAChRs, we examined if the complete genetic deletion of adenosine A(2A)Rs in mice induces compensatory alterations in the binding of different nAChR subtypes, and whether the long-term effects of nicotine on nAChR regulation are altered in the absence of the A(2A)R gene. Quantitative autoradiography was used to measure cytisine-sensitive [¹²⁵I]epibatidine and [¹²⁵I]α-bungarotoxin binding to α4β2* and α7 nAChRs, respectively, in brain sections of drug-naïve (n = 6) or nicotine treated (n = 5-7), wild-type and adenosine A(2A)R knockout mice. Saline or nicotine (7.8 mg/kg/day; free-base weight) were administered to male CD1 mice via subcutaneous osmotic minipumps for a period of 14 days. Blood plasma levels of nicotine and cotinine were measured at the end of treatment. There were no compensatory developmental alterations in nAChR subtype distribution or density in drug-naïve A(2A)R knockout mice. In nicotine treated wild-type mice, both α4β2* and α7 nAChR binding sites were increased compared with saline treated controls. The genetic ablation of adenosine A(2A)Rs prevented nicotine-induced upregulation of α7 nAChRs, without affecting α4β2* receptor upregulation. This selective effect was observed at plasma levels of nicotine that were within the range reported for smokers (10-50 ng ml⁻¹). Our data highlight the involvement of adenosine A(2A)Rs in the mechanisms of nicotine-induced α7 nAChR upregulation, and identify A(2A)Rs as novel pharmacological targets for modulating the long-term effects of nicotine on α7 receptors.

  12. Role and Function of A2A and A₃ Adenosine Receptors in Patients with Ankylosing Spondylitis, Psoriatic Arthritis and Rheumatoid Arthritis.

    PubMed

    Ravani, Annalisa; Vincenzi, Fabrizio; Bortoluzzi, Alessandra; Padovan, Melissa; Pasquini, Silvia; Gessi, Stefania; Merighi, Stefania; Borea, Pier Andrea; Govoni, Marcello; Varani, Katia

    2017-03-24

    Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are chronic inflammatory rheumatic diseases that affect joints, causing debilitating pain and disability. Adenosine receptors (ARs) play a key role in the mechanism of inflammation, and the activation of A2A and A₃AR subtypes is often associated with a reduction of the inflammatory status. The aim of this study was to investigate the involvement of ARs in patients suffering from early-RA (ERA), RA, AS and PsA. Messenger RNA (mRNA) analysis and saturation binding experiments indicated an upregulation of A2A and A₃ARs in lymphocytes obtained from patients when compared with healthy subjects. A2A and A₃AR agonists inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation and reduced inflammatory cytokines release, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. Moreover, A2A and A₃AR activation mediated a reduction of metalloproteinases (MMP)-1 and MMP-3. The effect of the agonists was abrogated by selective antagonists demonstrating the direct involvement of these receptor subtypes. Taken together, these data confirmed the involvement of ARs in chronic autoimmune rheumatic diseases highlighting the possibility to exploit A2A and A₃ARs as therapeutic targets, with the aim to limit the inflammatory responses usually associated with RA, AS and PsA.

  13. Interaction between serotonin 5-HT2A receptor gene and dopamine transporter (DAT1) gene polymorphisms influences personality trait of persistence in Austrian Caucasians.

    PubMed

    Schosser, Alexandra; Fuchs, Karoline; Scharl, Theresa; Schloegelhofer, Monika; Kindler, Jochen; Mossaheb, Nilufar; Kaufmann, Rainer M; Leisch, Friedrich; Kasper, Siegfried; Sieghart, Werner; Aschauer, Harald N

    2010-03-01

    We examined 89 normal volunteers using Cloninger's Temperament and Character Inventory (TCI). Genotyping the 102T/C polymorphism of the serotonin 5HT2A receptor gene and the ser9gly polymorphism in exon 1 of the dopamine D3 receptor (DRD3) gene was performed using PCR-RFLP, whereas the dopamine transporter (DAT1) gene variable number of tandem repeats (VNTR) polymorphism was investigated using PCR amplification followed by electrophoresis in an 8% acrylamide gel with a set of size markers. We found a nominally significant association between gender and harm avoidance (P=0.017; women showing higher scores). There was no association of either DAT1, DRD3 or 5HT2A alleles or genotypes with any dimension of the TCI applying Kruskal-Wallis rank-sum tests. Comparing homozygote and heterozygote DAT1 genotypes, we found higher novelty seeking scores in homozygotes (P=0.054). We further found a nominally significant interaction between DAT1 and 5HT2A homo-/heterozygous gene variants (P=0.0071; DAT1 and 5HT2A genotypes P value of 0.05), performing multivariate analysis of variance (MANOVA). Examining the temperamental TCI subscales, this interaction was associated with persistence (genotypes: P=0.004; homo-/heterozygous gene variants: P=0.0004). We conclude that an interaction between DAT1 and 5HT2A genes might influence the temperamental personality trait persistence.

  14. The role of adenosine A1 and A2A receptors in the caffeine effect on MDMA-induced DA and 5-HT release in the mouse striatum.

    PubMed

    Górska, A M; Gołembiowska, K

    2015-04-01

    3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") popular as a designer drug is often used with caffeine to gain a stronger stimulant effect. MDMA induces 5-HT and DA release by interaction with monoamine transporters. Co-administration of caffeine and MDMA may aggravate MDMA-induced toxic effects on DA and 5-HT terminals. In the present study, we determined whether caffeine influences DA and 5-HT release induced by MDMA. We also tried to find out if adenosine A1 and A2A receptors play a role in the effect of caffeine by investigating the effect of the selective adenosine A1 and A2A receptor antagonists, DPCPX and KW 6002 on DA and 5-HT release induced by MDMA. Mice were treated with caffeine (10 mg/kg) and MDMA (20 or 40 mg/kg) alone or in combination. DA and 5-HT release in the mouse striatum was measured using in vivo microdialysis. Caffeine exacerbated the effect of MDMA on DA and 5-HT release. DPCPX or KW 6002 co-administered with MDMA had similar influence as caffeine, but KW 6002 was more potent than caffeine or DPCPX. To exclude the contribution of MAO inhibition by caffeine in the caffeine effect on MDMA-induced increase in DA and 5-HT, we also tested the effect of the nonxanthine adenosine receptor antagonist CGS 15943A lacking properties of MAO activity modification. Our findings indicate that adenosine A1 and A2A receptor blockade may account for the caffeine-induced exacerbation of the MDMA effect on DA and 5-HT release and may aggravate MDMA toxicity.

  15. Gamma aminobutyric acid B and 5-hydroxy tryptamine 2A receptors functional regulation during enhanced liver cell proliferation by GABA and 5-HT chitosan nanoparticles treatment.

    PubMed

    Shilpa, Joy; Pretty, Mary Abraham; Anitha, Malat; Paulose, Cheramadathikudyil Skaria

    2013-09-05

    Liver is one of the major organs in vertebrates and hepatocytes are damaged by many factors. The liver cell maintenance and multiplication after injury and treatment gained immense interest. The present study investigated the role of Gamma aminobutyric acid (GABA) and serotonin or 5-hydroxytryptamine (5-HT) coupled with chitosan nanoparticles in the functional regulation of Gamma aminobutyric acid B and 5-hydroxy tryptamine 2A receptors mediated cell signaling mechanisms, extend of DNA methylation and superoxide dismutase activity during enhanced liver cell proliferation. Liver injury was achieved by partial hepatectomy of male Wistar rats and the GABA and 5-HT chitosan nanoparticles treatments were given intraperitoneally. The experimental groups were sham operated control (C), partially hepatectomised rats with no treatment (PHNT), partially hepatectomised rats with GABA chitosan nanoparticle (GCNP), 5-HT chitosan nanoparticle (SCNP) and a combination of GABA and 5-HT chitosan nanoparticle (GSCNP) treatments. In GABA and 5-HT chitosan nanoparticle treated group there was a significant decrease (P<0.001) in the receptor expression of Gamma aminobutyric acid B and a significant increase (P<0.001) in the receptor expression of 5-hydroxy tryptamine 2A when compared to PHNT. The cyclic adenosine monophosphate content and its regulatory protein, presence of methylated DNA and superoxide dismutase activity were decreased in GCNP, SCNP and GSCNP when compared to PHNT. The Gamma aminobutyric acid B and 5-hydroxy tryptamine 2A receptors coupled signaling elements played an important role in GABA and 5-HT chitosan nanoparticles induced liver cell proliferation which has therapeutic significance in liver disease management.

  16. The alpha2 adrenergic receptor antagonist idazoxan, but not the serotonin-2A receptor antagonist M100907, partially attenuated reward deficits associated with nicotine, but not amphetamine, withdrawal in rats.

    PubMed

    Semenova, Svetlana; Markou, Athina

    2010-10-01

    Based on phenomenological similarities between anhedonia (reward deficits) associated with drug withdrawal and the negative symptoms of schizophrenia, we showed previously that the atypical antipsychotic clozapine attenuated reward deficits associated with psychostimulant withdrawal. Antagonism of alpha(2) adrenergic and 5-HT(2A) receptors may contribute to these effects of clozapine. We investigated here whether blockade of alpha(2) or 5-HT(2A) receptors by idazoxan and M100907, respectively, would reverse anhedonic aspects of psychostimulant withdrawal. Idazoxan treatment facilitated recovery from spontaneous nicotine, but not amphetamine, withdrawal by attenuating reward deficits and increase the number of somatic signs. Thus, alpha(2) adrenoceptor blockade may have beneficial effects against nicotine withdrawal and may be involved in the effects of clozapine previously observed. M100907 worsened the anhedonia associated with nicotine and amphetamine withdrawal, suggesting that monotherapy with M100907 may exacerbate the expression of the negative symptoms of schizophrenia or nicotine withdrawal symptoms in people, including schizophrenia patients, attempting to quit smoking.

  17. Differential regulation of HIF-1α and HIF-2α in neuroblastoma: Estrogen-related receptor alpha (ERRα) regulates HIF2A transcription and correlates to poor outcome

    SciTech Connect

    Hamidian, Arash; Stedingk, Kristoffer von; Munksgaard Thorén, Matilda; Mohlin, Sofie; Påhlman, Sven

    2015-06-05

    Hypoxia-inducible factors (HIFs) are differentially regulated in tumor cells. While the current paradigm supports post-translational regulation of the HIF-α subunits, we recently showed that hypoxic HIF-2α is also transcriptionally regulated via insulin-like growth factor (IGF)-II in the childhood tumor neuroblastoma. Here, we demonstrate that transcriptional regulation of HIF-2α seems to be restricted to neural cell-derived tumors, while HIF-1α is canonically regulated at the post-translational level uniformly across different tumor forms. Enhanced expression of HIF2A mRNA at hypoxia is due to de novo transcription rather than increased mRNA stability, and chemical stabilization of the HIF-α proteins at oxygen-rich conditions unexpectedly leads to increased HIF2A transcription. The enhanced HIF2A levels do not seem to be dependent on active HIF-1. Using a transcriptome array approach, we identified members of the Peroxisome proliferator-activated receptor gamma coactivator (PGC)/Estrogen-related receptor (ERR) complex families as potential regulators of HIF2A. Knockdown or inhibition of one of the members, ERRα, leads to decreased expression of HIF2A, and high expression of the ERRα gene ESRRA correlates with poor overall and progression-free survival in a clinical neuroblastoma material consisting of 88 tumors. Thus, targeting of ERRα and pathways regulating transcriptional HIF-2α are promising therapeutic avenues in neuroblastoma. - Highlights: • Transcriptional control of HIF-2α is restricted to neural cell-derived tumors. • Enhanced transcription of HIF2A is not due to increased mRNA stability. • Chemical stabilization of the HIF-α subunits leads to increased HIF2A transcription. • ERRα regulates HIF2A mRNA expression in neuroblastoma. • High expression of ESRRA correlates to poor outcome in neuroblastoma.

  18. Metaplasticity gated through differential regulation of GluN2A versus GluN2B receptors by Src family kinases

    PubMed Central

    Yang, Kai; Trepanier, Catherine; Sidhu, Bikram; Xie, Yu-Feng; Li, Hongbin; Lei, Gang; Salter, Michael W; Orser, Beverley A; Nakazawa, Takanobu; Yamamoto, Tadashi; Jackson, Michael F; MacDonald, John F

    2012-01-01

    Metaplasticity is a higher form of synaptic plasticity that is essential for learning and memory, but its molecular mechanisms remain poorly understood. Here, we report that metaplasticity of transmission at CA1 synapses in the hippocampus is mediated by Src family kinase regulation of NMDA receptors (NMDARs). We found that stimulation of G-protein-coupled receptors (GPCRs) regulated the absolute contribution of GluN2A-versus GluN2B-containing NMDARs in CA1 neurons: pituitary adenylate cyclase activating peptide 1 receptors (PAC1Rs) selectively recruited Src kinase, phosphorylated GluN2ARs, and enhanced their functional contribution; dopamine 1 receptors (D1Rs) selectively stimulated Fyn kinase, phosphorylated GluN2BRs, and enhanced these currents. Surprisingly, PAC1R lowered the threshold for long-term potentiation while long-term depression was enhanced by D1R. We conclude that metaplasticity is gated by the activity of GPCRs, which selectively target subtypes of NMDARs via Src kinases. PMID:22187052

  19. The 2.6 Angstrom Crystal Structure of a Human A[subscript 2A] Adenosine Receptor Bound to an Antagonist

    SciTech Connect

    Jaakola, Veli-Pekka; Griffith, Mark T.; Hanson, Michael A.; Cherezov, Vadim; Chien, Ellen Y.T.; Lane, J. Robert; IJzerman, Adriaan P.; Stevens, Raymond C.

    2009-01-15

    The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A{sub 2A} adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.

  20. Cross-talk from β-adrenergic receptors modulates α2A-adrenergic receptor endocytosis in sympathetic neurons via protein kinase A and spinophilin.

    PubMed

    Cottingham, Christopher; Lu, Roujian; Jiao, Kai; Wang, Qin

    2013-10-04

    Inter-regulation of adrenergic receptors (ARs) via cross-talk is a long appreciated but mechanistically unclear physiological phenomenon. Evidence from the AR literature and our own extensive studies on regulation of α2AARs by the scaffolding protein spinophilin have illuminated a potential novel mechanism for cross-talk from β to α2ARs. In the present study, we have characterized a mode of endogenous AR cross-talk in native adrenergic neurons whereby canonical βAR-mediated signaling modulates spinophilin-regulated α2AAR endocytosis through PKA. Our findings demonstrate that co-activation of β and α2AARs, either by application of endogenous agonist or by simultaneous stimulation with distinct selective agonists, results in acceleration of endogenous α2AAR endocytosis in native neurons. We show that receptor-independent PKA activation by forskolin is sufficient to accelerate α2AAR endocytosis and that α2AAR stimulation alone drives accelerated endocytosis in spinophilin-null neurons. Endocytic response acceleration by β/α2AAR co-activation is blocked by PKA inhibition and lost in spinophilin-null neurons, consistent with our previous finding that spinophilin is a substrate for phosphorylation by PKA that disrupts its interaction with α2AARs. Importantly, we show that α2AR agonist-mediated α2AAR/spinophilin interaction is blocked by βAR co-activation in a PKA-dependent fashion. We therefore propose a novel mechanism for cross-talk from β to α2ARs, whereby canonical βAR-mediated signaling coupled to PKA activation results in phosphorylation of spinophilin, disrupting its interaction with α2AARs and accelerating α2AAR endocytic responses. This mechanism of cross-talk has significant implications for endogenous adrenergic physiology and for therapeutic targeting of β and α2AARs.

  1. Contribution of a helix 5 locus to selectivity of hallucinogenic and nonhallucinogenic ligands for the human 5-hydroxytryptamine2A and 5-hydroxytryptamine2C receptors: direct and indirect effects on ligand affinity mediated by the same locus.

    PubMed

    Almaula, N; Ebersole, B J; Ballesteros, J A; Weinstein, H; Sealfon, S C

    1996-07-01

    An important determinant of the neurobehavioral responses induced by a drug is its relative receptor selectivity. The molecular basis of ligand selectivity of hallucinogenic and nonhallucinogenic compounds of varying structural classes for the human 5-hydroxytryptamine (5-HT)2A and 5-HT2C receptors was investigated with the use of reciprocal site-directed mutagenesis. Because these two closely related receptor subtypes differ in the amino acid present at position 5.46 (residues 242 and 222 in the sequences, respectively), the effects of corresponding substitutions in the 5-HT2A[S5.46(242)-->A] and 5-HT2C[A5.46(222)-->S] receptors were studied in tandem. By studying both receptors, the direct and indirect effects of mutations on affinity and selectivity can be distinguished. The ergolines studied, mesulergine (selective for the 5-HT2C receptor) and d-lysergic acid diethylamide (selective for the 5-HT2A receptor), reversed their relative affinity with mutations in each receptor, supporting a direct role of this locus in the selectivity of these ligands. However, interchange mutations in either receptor led to decreased or unchanged affinity for (+/-)-1-)(2,5-dimethoxy-4-iodophenyl)-2-aminopropane and ketanserin, which have higher affinity for the 5-HT2A receptor, consistent with little contribution of this locus to the selectivity of these ligands. The indoleamines studied were affected differently by mutations in each receptor, suggesting that they bind differently to the two receptor subtypes. Mutation of this locus in the 5-HT2A receptor decreased the affinity of all indoleamines, whereas the interchange mutation of the 5-HT2C receptor did not affect indoleamine affinity. These results are consistent with a direct interaction between this side chain and indoleamines for the 5-HT2A receptor but not for the 5-HT2C receptor. Furthermore, this analysis shows that the higher affinity of 5-HT and tryptamine for the 5-HT2C receptor than for the 5-HT2A receptors is not

  2. A(2A) adenosine receptors are differentially modulated by pharmacological treatments in rheumatoid arthritis patients and their stimulation ameliorates adjuvant-induced arthritis in rats.

    PubMed

    Vincenzi, Fabrizio; Padovan, Melissa; Targa, Martina; Corciulo, Carmen; Giacuzzo, Sarah; Merighi, Stefania; Gessi, Stefania; Govoni, Marcello; Borea, Pier Andrea; Varani, Katia

    2013-01-01

    A(2A) adenosine receptors (ARs) play a key role in the inhibition of the inflammatory process. The purpose of this study was to evaluate the modulation of A(2A)ARs in rheumatoid arthritis (RA) patients after different pharmacological treatments and to investigate the effect of A(2A)AR stimulation in a rat model of arthritis. We investigated A(2A)AR density and functionality in RA progression by using a longitudinal study in RA patients before and after methotrexate (MTX), anti-TNFα agents or rituximab treatments. A(2A)ARs were analyzed by saturation binding assays in lymphocytes from RA patients throughout the 24-month study timeframe. In an adjuvant-induced arthritis model in rats we showed the efficacy of the A(2A)AR agonist, CGS 21680 in comparison with standard therapies by means of paw volume assessment, radiographic and ultrasonographic imaging. Arthritic-associated pain was investigated in mechanical allodynia and thermal hyperalgesia tests. IL-10 release following A(2A)AR stimulation in lymphocytes from RA patients and in serum from arthritic rats was measured. In lymphocytes obtained from RA patients, the A(2A)AR up-regulation was gradually reduced in function of the treatment time and the stimulation of these receptors mediated a significant increase of IL-10 production. In the same cells, CGS 21680 did not affected cell viability and did not produced cytotoxic effects. The A(2A)AR agonist CGS 21680 was highly effective, as suggested by the marked reduction of clinical signs, in rat adjuvant-induced arthritis and associated pain. This study highlighted that A(2A)AR agonists represent a physiological-like therapeutic alternative for RA treatment as suggested by the anti-inflammatory role of A(2A)ARs in lymphocytes from RA patients. The effectiveness of A(2A)AR stimulation in a rat model of arthritis supported the role of A(2A)AR agonists as potential pharmacological treatment for RA.

  3. Tolerance to LSD and DOB induced shaking behaviour: differential adaptations of frontocortical 5-HT(2A) and glutamate receptor binding sites.

    PubMed

    Buchborn, Tobias; Schröder, Helmut; Dieterich, Daniela C; Grecksch, Gisela; Höllt, Volker

    2015-03-15

    Serotonergic hallucinogens, such as lysergic acid diethylamide (LSD) and dimethoxy-bromoamphetamine (DOB), provoke stereotype-like shaking behaviour in rodents, which is hypothesised to engage frontocortical glutamate receptor activation secondary to serotonin2A (5-HT2A) related glutamate release. Challenging this hypothesis, we here investigate whether tolerance to LSD and DOB correlates with frontocortical adaptations of 5-HT2A and/or overall-glutamate binding sites. LSD and DOB (0.025 and 0.25 mg/kg, i.p.) induce a ketanserin-sensitive (0.5 mg/kg, i.p., 30-min pretreatment) increase in shaking behaviour (including head twitches and wet dog shakes), which with repeated application (7× in 4 ds) is undermined by tolerance. Tolerance to DOB, as indexed by DOB-sensitive [(3)H]spiroperidol and DOB induced [(35)S]GTP-gamma-S binding, is accompanied by a frontocortical decrease in 5-HT2A binding sites and 5-HT2 signalling, respectively; glutamate-sensitive [(3)H]glutamate binding sites, in contrast, remain unchanged. As to LSD, 5-HT2 signalling and 5-HT2A binding, respectively, are not or only marginally affected, yet [(3)H]glutamate binding is significantly decreased. Correlation analysis interrelates tolerance to DOB to the reduced 5-HT2A (r=.80) as well as the unchanged [(3)H]glutamate binding sites (r=.84); tolerance to LSD, as opposed, shares variance with the reduction in [(3)H]glutamate binding sites only (r=.86). Given that DOB and LSD both induce tolerance, one correlating with 5-HT2A, the other with glutamate receptor adaptations, it might be inferred that tolerance can arise at either level. That is, if a hallucinogen (like LSD in our study) fails to induce 5-HT2A (down-)regulation, glutamate receptors (activated postsynaptic to 5-HT2A related glutamate release) might instead adapt and thus prevent further overstimulation of the cortex.

  4. In vivo assessment of coronary flow and cardiac function after bolus adenosine injection in adenosine receptor knockout mice.

    PubMed

    Teng, Bunyen; Tilley, Stephen L; Ledent, Catherine; Mustafa, S Jamal

    2016-06-01

    Bolus injections of adenosine and the A2A adenosine receptor (AR) selective agonist (regadenoson) are used clinically as a substitute for a stress test in people who cannot exercise. Using isolated tissue preparations, our lab has shown that coronary flow and cardiac effects of adenosine are mostly regulated by the AR subtypes A1, A2A, and A2B In this study, we used ultrasound imaging to measure the in vivo effects of adenosine on coronary blood flow (left coronary artery) and cardiac function in anesthetized wild-type, A1 knockout (KO), A2AKO, A2BKO, A3KO, A1, and A3 double KO (A1/3 DKO) and A2A and A2B double KO (A2A/2B DKO) mice in real time. Echocardiographic and Doppler studies were performed using a Visualsonic Vevo 2100 ultrasound system. Coronary blood flow (CBF) baseline data were obtained when animals were anesthetized with 1% isoflourane. Diameter (D) and velocity time integral (VTI) were measured on the left coronary arteries (CBF = ((π/4) × D(2) × VTI × HR)/1000). CBF changes were the highest within 2 min of injection (about 10 mg/kg). Heart rate, cardiac output, and stroke volume were measured by tracing the left ventricle long axis. Our data support a role for the A2 AR in CBF and further support our conclusions of previous studies from isolated tissues. Adenosine-mediated decreases in cardiac output and stroke volume may be A2B and/or A3 AR-mediated; however, the A1 and A2 ARs also play roles in overall cardiac function. These data further provide a powerful translational tool in studying the cardiovascular effects of adenosine in disease states.

  5. In search of novel ligands using a structure-based approach: a case study on the adenosine A2A receptor

    NASA Astrophysics Data System (ADS)

    Lenselink, Eelke B.; Beuming, Thijs; van Veen, Corine; Massink, Arnault; Sherman, Woody; van Vlijmen, Herman W. T.; IJzerman, Adriaan P.

    2016-10-01

    In this work, we present a case study to explore the challenges associated with finding novel molecules for a receptor that has been studied in depth and has a wealth of chemical information available. Specifically, we apply a previously described protocol that incorporates explicit water molecules in the ligand binding site to prospectively screen over 2.5 million drug-like and lead-like compounds from the commercially available eMolecules database in search of novel binders to the adenosine A2A receptor (A2AAR). A total of seventy-one compounds were selected for purchase and biochemical assaying based on high ligand efficiency and high novelty (Tanimoto coefficient ≤0.25 to any A2AAR tested compound). These molecules were then tested for their affinity to the adenosine A2A receptor in a radioligand binding assay. We identified two hits that fulfilled the criterion of 50 % radioligand displacement at a concentration of 10 μM. Next we selected an additional eight novel molecules that were predicted to make a bidentate interaction with Asn2536.55, a key interacting residue in the binding pocket of the A2AAR. None of these eight molecules were found to be active. Based on these results we discuss the advantages of structure-based methods and the challenges associated with finding chemically novel molecules for well-explored targets.

  6. Interleukin-1beta but not tumor necrosis factor-alpha potentiates neuronal damage by quinolinic acid: protection by an adenosine A2A receptor antagonist.

    PubMed

    Stone, Trevor W; Behan, Wilhelmina M H

    2007-04-01

    Quinolinic acid is an agonist at glutamate receptors sensitive to N-methyl-D-aspartate (NMDA). It has been implicated in neural dysfunction associated with infections, trauma, and ischemia, although its neurotoxic potency is relatively low. This study was designed to examine the effects of a combination of quinolinic acid and the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). Compounds were administered to the hippocampus of anesthetized male rats, animals being allowed to recover for 7 days before histological analysis of the hippocampus for neuronal damage estimated by counting of intact, healthy neurons. A low dose of quinolinic acid or IL-1beta produced no damag